Measuring fluorescence-lifetime and bio-impedance sensors for cell based assays using a network analyzer integrated circuit.

Cell culture assays for therapeutic drug screening today are fully automated. Vitality of the cells is monitored by different sensors. For such a system, we propose a new reader unit, which is capable of reading two different fluorescent sensors and electrical impedance in 24-well-plates. Main goals are to reduce cost, complexity and size while achieving a similar performance as the existing reader unit. To achieve this, measurement electronics and signal paths for frequency domain fluorescence and bio-impedance measurement are combined. Central component is an integrated circuit for impedance spectroscopy. A new compact and economic optical setup is developed to read two different sensor spots on the bottom of the well. Measurement errors introduced by different components like DFT leakage, and frequency dependent signal delays are evaluated and compensated. A set of commercially available fluorescence sensor spots is used to verify the read out performance. The results are usable, with noise slightly higher than commercial readers. To verify the impedance measurement accuracy, measurements of known resistances are conducted. In the relevant impedance and frequency range for biological applications a suitable accuracy is achieved. Due to the higher sampling rate of the new reader, the higher noise can be reduced through averaging. The new system is significantly smaller and cheaper to manufacture than commercially available devices.

[Medicine 4.0: examples of applications of electronics, information technology and microsystems in modern medicine]. / « Médecine 4.0 ¼ - Exemples d'application de l'électronique, de la technologie de l'information et des microsystèmes dans la médecine moderne.

The electronic advances of the last hundred years have made enormous contributions to medical research and the development of new therapeutic methods. In recent years in particular, it has been demonstrated that intelligent sensors, with appropriate radio interfaces, will soon allow diagnostic and therapeutic processes in medicine to be linked to one another - this will enable the development of completely new forms of therapy [1]. This new "Medicine 4.0" was the subject of a first article in the series, which presented the progress achieved through the merging of microsensor technology, microelectronics, information and communication technologies, with a particular focus on the case of personalized chemotherapy. The purpose of this new article is to present more practical applications of these new therapeutic methods.

Electronics for better healthcare.

Microelectronics and microsystem technology have changed our daily lives considerably in the past 50 years. Countless everyday objects contain microelectronic components. In healthcare up to the present, however, it has not been possible to make major alterations in introducing electronics and information technology that would lead to innovative improvements and greater transparency. This paper describes initial steps in diagnostics and oncological therapy including telematic healthcare systems which can, for example, assist patients with cardiovascular diseases and shows, through these areas, how electronics and microsystems technology can contribute to better healthcare.

Determination of dynamic doxorubicin-EC50 value in an automated high-content workstation for cellular assays.

To overcome the problems of endpoint tests routinely required for EC50 determination, we utilized a novel automated high-content workstation and calculated a time-resolved EC50 value of MCF-7 mamma carcinoma cells treated with a pharmacologic agent. Measuring parameters were the cellular oxygen consumption and the extracellular acidification. These parameters were detected in real-time and label free with optochemical sensor spots in a modified 24-well sensor plate. In particular, the objective was to compare the measuring data of the workstation with a classical standard resazurin cell assay and to transfer the benefit of continuously recorded metabolic data of the workstation to practical time-resolved information about the effect of the applied active reagent (doxorubicin). MCF-7 cells were treated with a broad range of doxorubicin concentrations (100 µM to 1 nM) over 24 h and cellular activities were investigated with both, the resazurin assay and the automated workstation. Twenty-four hours after treatment, the resazurin assay showed an EC50 value (6.3 µM) which was about one decade above the value obtained from oxygen consumption rate (0.37 µM) and extracellular acidification rate (0.72 µM), measured with the workstation. Presumably, the differences are due to the different metabolic nature and regulation behind these measuring parameters. By polynomial fitting of continuously recorded metabolic data, we were able to point out a dynamic, time-resolved EC50 characteristic for different time points. The workstation is a powerful tool to record in vitro kinetic data of pharmacologic effects in vital cells in an automated experimental run.

Comparison of established and novel purity tests for the quality control of heparin by means of a set of 177 heparin samples.

The widespread occurrence of heparin contaminated with oversulfated chrondroitin sulfate (OSCS) in 2008 initiated a comprehensive revision process of the Pharmacopoeial heparin monographs and stimulated research in analytical techniques for the quality control of heparin. Here, a set of 177 heparin samples from the market in 2008 as well as pure heparin sodium spiked with defined amounts of OSCS and DS were used to evaluate established and novel methods for the quality control of heparin. Besides (1)H nuclear magnetic resonance spectroscopy (NMR), the assessment included two further spectroscopic methods, i.e., attenuated total reflection-infrared spectroscopy (ATR-IR) and Raman spectroscopy, three coagulation assays, i.e., activated partial thromboplastin time (aPTT) performed with both sheep and human plasma and the prothrombin time (PT), and finally two novel purity assays, each consisting of an incubation step with heparinase I followed by either a fluorescence measurement (Inc-PolyH-assay) or by a chromogenic aXa-assay (Inc-aXa-assay). NMR was shown to allow not only sensitive detection, but also quantification of OSCS by using the peak-height method and a response factor determined by calibration. Chemometric evaluation of the NMR, ATR-IR, and Raman spectra by statistical classification techniques turned out to be best with NMR spectra concerning the detection of OSCS. The validity of the aPTT, the current EP assay, could be considerably improved by replacing the sheep plasma by human plasma. In this way, most of the contaminated heparin samples did not meet the novel potency limit of 180 IU/mg. However, also more than 50% of the uncontaminated samples had <180 IU/MG. In contrast to the aPTT, the PT specifically detects OSCS and other heparin mimetics (LOD 3%). About ten times more sensitive are both the Inc-PolyH-assay and the Inc-aXa-assay, two rapid and simple quantification assays for heparin mimetics. The determined OSCS contents of the heparin samples excellently correlated with those calculated from the NMR spectra. In conclusion, NMR proved to be the current spectroscopic method of choice. The two two-step-assays represent options to supplement NMR, especially as tests for the initial screening, since they detect any heparin mimetic without requiring special expertise for interpretation of the results.

Magnetic stimulation and depression of mammalian networks in primary neuronal cell cultures.

For transcranial magnetic stimulation (TMS), the coupling of induced electric fields with neurons in gray matter is not well understood. There is little information on optimal stimulation parameters and on basic cellular mechanisms. For this reason, magnetic stimulation of spontaneously active neuronal networks, grown on microelectrode arrays in culture, was employed as a test environment. This allowed use of smaller coils and the continual monitoring of network action potential (AP) activity before, during, and for long periods after stimulation. Biphasic, rectangular, and 500 micros long pulses were used at mean pulse frequencies (MPFs) ranging from 3 to 100 Hz on both spinal cord (SC) and frontal cortex (FC) cultures. Contrary to stimulation of organized fiber bundles, APs were not elicited directly. Responses were predominantly inhibitory, dose dependent, with onset times between 10 s and several minutes. Spinal networks showed a greater sensitivity to activity suppression. Under pharmacological disinhibition, some excitation was seen at low pulse frequencies. FC cultures showed greater excitatory responses than SC networks. The observed primary inhibitory responses imply interference with synaptic exocytosis mechanisms. With 20,000 pulses at 10 Hz, 40% inhibition was maintained for over 30 min with full recovery, suggesting possible application to nonchemical, noninvasive pain management.

Effects of Basella alba and Hibiscus macranthus extracts on testosterone production of adult rat and bull Leydig cells.

AIM: To determine the androgenic effects of Basella alba and Hibiscus macranthus extracts in the rat and the bull, and to develop a novel in vitro test system using Leydig cells from bull testes. METHODS: The effect of methanol extracts from both plants on testosterone production in isolated Leydig cells from the rat and the bull was analyzed using 125I-radioimmunoassay (125I-RIA). Rat Leydig cells were obtained by common methods, whereas a novel technique was used to purify Leydig cells from bull testes. RESULTS: Bull testes from the slaughter house were a cheap source of pure Leydig cells. In culture, these cells produced testosterone for 5-6 days, which can be stimulated by human chorionic gonadotrophin (hCG). Basella alba extracts significantly enhanced testosterone production in bull and rat Leydig cells in a concentration-dependent manner. Hibiscus macranthus showed no androgenic effect but was shown to inhibit testosterone production at higher concentrations. CONCLUSION: Leydig cells purified from bull testes can be used as an alternative tool in experimental animal research. Certain fractions of Basella alba extract demonstrated androgenic potential whereas Hibiscus macranthus extracts did not.

[Stress and psoriasis -- a psychoneuroimmunological study]. / Stress und Psoriasis -- eine psychoneuroimmunologische Studie.

UNLABELLED: Psoriasis is a polygenetic hereditary multifactorial disease which may be influenced by a number of environmental factors. To date only a few studies experimentally investigated the influence of stress on psoriasis. One problem of these studies is that it remains unclear whether the experimental findings are relevant for the entire group of patients, or whether there are subgroups who are particularly susceptible to stress. Therefore our main objective is to examine whether experimental stressors can identify subgroups of patients who are particularly susceptible to stress and if these differ in immunological parameters. METHOD: The Trier Social Stress Test (TSST) was used as stressor. The severity was recorded both objectively using the PASI (Psoriasis Area and Severity Index) as well as subjectively by the patient. Somatic parameters for which stress reactivity is known but no direct relationship to psoriasis is assumed were selected and exploratively examined within the present study (salivary cortisol). The second set of parameters for which the stress reactivity was unclear included variables which are linked to the pathophysiology and/or the severity of psoriasis. In addition to salivary cortisol, eosinophils, ICAM-3, and sIL-2R were determined in serum. 38 psoriasis patients and 38 control subjects were examined (21 male and 17 female participants within each group). RESULTS: The PASI correlated very inconsistently with the subjective severity parameters. The relationships between severity and blood parameters tested showed a systematic relationship for eosinophils only. The TSST is suitable for eliciting stress in psoriasis patients. In one subgroup, there was an increase in skin affliction, while skin affliction in the second group remained constant or decreased. A classification into stress-reactive or non-reactive patients cannot, however, be supported by the immunological parameters tested.

Effects of Basella alba and Hibiscus macranthus extracts on testosterone production of adult rat and bull Leydig cells / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To determine the androgenic effects of Basella alba and Hibiscus macranthus extracts in the rat and the bull, and to develop a novel in vitro test system using Leydig cells from bull testes.</p><p><b>METHODS</b>The effect of methanol extracts from both plants on testosterone production in isolated Leydig cells from the rat and the bull was analyzed using 125I-radioimmunoassay (125I-RIA). Rat Leydig cells were obtained by common methods, whereas a novel technique was used to purify Leydig cells from bull testes.</p><p><b>RESULTS</b>Bull testes from the slaughter house were a cheap source of pure Leydig cells. In culture, these cells produced testosterone for 5-6 days, which can be stimulated by human chorionic gonadotrophin (hCG). Basella alba extracts significantly enhanced testosterone production in bull and rat Leydig cells in a concentration-dependent manner. Hibiscus macranthus showed no androgenic effect but was shown to inhibit testosterone production at higher concentrations.</p><p><b>CONCLUSION</b>Leydig cells purified from bull testes can be used as an alternative tool in experimental animal research. Certain fractions of Basella alba extract demonstrated androgenic potential whereas Hibiscus macranthus extracts did not.</p>

Interdisciplinary diagnostics in environmental medicine--findings and follow-up in patients with chronic medically unexplained health complaints.

PROBLEM: In patients attributing their chronic, medically unexplained complaints to environmental factors the greatest challenge is to overcome their disabling belief in toxicogenic explanations. METHOD: Patients presenting with health complaints that they attributed to environmental causes in an environmental outpatient department (EOPD) within a university medical center in Germany were studied. An interdisciplinary review of previously diagnosed medical conditions, current clinical consultations, personal risk communication and therapeutic advice is presented. Additionally, patient contentedness, complaint development, and belief in environmental attribution in a follow-up interview are given. RESULTS: The open, prospective study comprises 51 patients reporting more than one complaint. Symptoms had lasted for more than 3 years in 63% of the cases. Seventy percent attributed their complaints to more than one environmental cause. The clinical diagnostic procedure reduced the number of prediagnosed clinical conditions by 50%. Numerous foregoing environmental laboratory analyses had overestimated toxicologically relevant findings. These were not confirmed in 80% (8/10) of the cases. In 8% (n = 4) of the patients a relevant environmental or occupational medical condition was found. A mental or behavioral condition was not considered to have first priority in explaining all complaints in 43% (22/51) of the patients. Among these, mostly respiratory or skin-related diseases were found. All patients contacted participated in a follow-up study after a minimum of 21 months. Sixty-seven percent reported having felt that they were taken seriously, 38% felt better after the beginning of the study, and 45% were no longer certain about the importance of the environmental attribution. Since 83% of the patients with a preceding residential diagnosis of MCS or SBS still believed in environmental causes of their complaints in the follow-up study, we conclude that these prediagnoses appear to be a risk for persisting attribution of the environmental factor. About one third (37%) of these patients with complaints that had not been medically explained by an organic condition during interdisciplinary diagnostics had meanwhile consulted a psychotherapist. CONCLUSIONS: Interdisciplinary diagnostics and scientifically based risk assessment in a specialized clinical center were effective and mostly well accepted by the patients and resulted in reduced attribution of complaints to environmental conditions. No indication was found that patients with complaints not medically explained by organic conditions were managed less successfully by this approach. Considering the high costs that these patients have previously caused, it appears valuable to apply an interdisciplinary diagnostic strategy.

A double-blind, randomized, placebo-controlled trial of n-3 versus n-6 fatty acid-based lipid infusion in atopic dermatitis.

BACKGROUND: In the involved epidermis of patients with atopic dermatitis, changes in the metabolism of eicosanoids with increased quantities of the arachidonic acid (AA)-derived lipoxygenase products have been observed. Free eicosapentaenoic acid (EPA), a fish oil-derived alternative (n-3) fatty acid, may compete with AA, resulting in an anti-inflammatory effect. METHODS: In a 10-day double-blind, randomized, placebo-controlled trial, 22 patients hospitalized for moderate-to-severe atopic dermatitis were randomly assigned to receive daily infusions of either a n-3 fatty acid-based lipid emulsion (fish oil, 10%; 200 mL/d) or a conventional n-6-lipid emulsion (soybean oil, 10%; 200 mL/d). Topical treatment was restricted to emollients. The severity of disease was evaluated daily with scoring of erythema, infiltration, and desquamation and by subjective patient scoring of clinical manifestations. In addition, plasma-free and total-bound fatty acids and the composition of membrane fatty acids in blood cells (thrombocytes, granulocytes, and erythrocytes), lipid mediators from isolated neutrophils and platelets, and lymphocyte-activation parameters were determined. RESULTS: Twenty patients completed the trial. Marked improvement from baseline was seen in both groups. On days 6, 7, 8, and 10, disease severity score-defined as the sum of all scores-was more pronounced (p < .05) in the n-3 group compared with the n-6 group. Free arachidonic acid in plasma did not change substantially in both groups, whereas plasma-free EPA, total-bound EPA, and the membrane EPA/AA ratio markedly increased in response to n-3-lipid infusion. In parallel, EPA-derived lipid mediators appeared, whereas lymphocyte functions were unaffected. In the post-treatment period (2/4 weeks), relapse was observed in some patients after n-3 psoralene-ultraviolet A (PUVA) infusion, whereas there was a marked long-term improvement in the n-6 group. CONCLUSIONS: IV n-3-fatty acid administration is effective in acutely improving the severity of atopic dermatitis, paralleled by changes in plasma and membrane fatty acid composition and lipid mediator synthesis. The long-term beneficial effects of IV n-6 fatty acids should be evaluated further.