Severe underquantification of HIV-1 group O isolates by major commercial PCR-based assays.

HIV-1 diversity poses major challenges to viral load assays because genetic polymorphisms can impede nucleic acid detection. In addition to the on-going viral diversification within the HIV-1 group M pandemic, HIV-1 genetic diversity is further increased by non-group M infections, such as HIV-1 groups O (HIV-1-O), N and P. We here conducted a systematic evaluation of commercially available PCR assays to detect HIV-1-O isolates. We collected 25 primary HIV-1-O isolates covering all genetic clusters within HIV-1-O. Subsequently, this panel of isolates was tested on eight commercially available quantitative and five qualitative HIV-1 PCR-based assays in serial dilutions. Sequence analyses were performed for severe cases of underquantification or lack of detection. We observed differences between the assays in quantification that depended on the HIV-1-O isolate's subgroup. All three tested HIV-1-O subgroup IV isolates were underquantified by the Roche CAP/CTM >800-fold compared to the Abbott RealTime assay. In contrast, the latter assay underquantified several subgroup I isolates >200-fold. Notably, the Xpert HIV-1 Viral Load test from Cepheid failed to detect two of the HIV-1-O isolates, whereas the Roche Cobas 8800 assay readily detected all isolates. Comparative sequence analyses identified polymorphisms in the HIV-1-O long-terminal repeat and integrase genes that likely underlie inadequate nucleic acid amplification. Potential viral load underquantification should be considered in therapeutic monitoring of HIV-1-O-infected patients. Pre-clinical assessments of HIV-1 diagnostic assays could be harmonized by establishing improved and internationally standardized panels of HIV-1 isolates that cover the dynamic diversity of circulating HIV-1 strains.

Variation analysis of six HCV viral load assays using low viremic HCV samples in the range of the clinical decision points for HCV protease inhibitors.

In the range of clinical decision points for response-guided therapy of HCV, there is still insufficient data concerning the conformity of quantification results obtained by different assays and their correlation with the HPS/CTM v2 assay which was used for initial clinical studies. In a head-to-head comparison, assay accuracy and detection rates of six quantitative assays [artus HCV QS-RGQ, COBAS Ampliprep/COBAS TaqMan HCV v1/v2, High Pure System/COBAS TaqMan (HPS), RealTime HCV, and Versant HCV1.0] were assessed by measuring WHO and PEI standards at dilution steps near clinical decision points. Detection rates and mean differences between assays were evaluated by analyzing twenty clinical samples at 10, 100, and 1,000 IU/mL. Ten replicates from specimens with different HCV genotypes were used to analyze pan-genotypic intra-assay variation. At ≤ 25 IU/mL, RealTime demonstrated the highest detection rates. With 0.1 log difference when testing clinical samples, results obtained from the Versant and RealTime assays matched best with results from HPS. Mean difference analysis across all assay results revealed wide differences between 0.01 and 0.75 log IU/mL. RealTime showed the lowest intra-assay variation across genotypes 1-4 (25, 100, 1,000 IU/mL). There are substantial analytical differences between viral load assays clinicians should be aware of. These variations may have impact on clinical decisions for patients on HCV triple therapy and may argue for assay-specific decision points equivalent to reference values established in studies using HPS. A comparison of quantification is recommended prior to a switch of assays during ongoing therapy.

Online monitoring of cellular metabolism in the MCF-7 carcinoma cell line treated with phytoestrogen extracts.

BACKGROUND: Phytoestrogens are naturally occurring, plant-derived, nonsteroidal phytochemicals with anticarcinogenic potential. The aim of this study was to isolate phytoestrogens from the flax root of Linum usitatissimum and to test their effect on cellular metabolism in the human mammalian carcinoma cell line MCF-7 using the Bionas 2500 analysis system. MATERIALS AND METHODS: Metabolically relevant parameters such as acidification, oxygen consumption and cell adhesion were registered continuously over 8 and 24 hours on six sensor chips in parallel at different concentrations of flax root extracts. RESULTS: The extracts from flax roots of L. usitatissimum reduced extracellular acidification, respiration and adhesion in a concentration-dependent manner. CONCLUSION: The Bionas 2500 analysis system allows multiparametric online monitoring of cellular processes and can be used to detect the mode of action of anticarcinogenic compounds in cellular metabolism.

[Direct costs for Parkinson's treatment in private neurology practices in Berlin]. / Direkte Kosten der Parkinson-Behandlung : Eine Erhebung in neurologischen Schwerpunktpraxen in Berlin.

BACKGROUND: Aim of this study was to assess the direct costs of Parkinson's disease (PD) within a 3-month period (i.e. the accounting period for the German statutory health insurance) in 12 neurological outpatient practices in Berlin during 2006. MATERIAL AND METHODS: A total of 425 patients (age 69.1+/-9.3 years, 185 females) were recruited, and sociodemographic and clinical data were obtained by a specific questionnaire. The distribution of costs was analyzed based on several clinical and patient parameters. The costs were calculated with different approaches: (1) prospectively, with the practices' accounting according to German uniform scales (GoA, EbM) and (2) retrospectively, with questionnaires for the Parkinson's patients. Costs were calculated according to current German guidelines of the statutory health insurance. Clinical parameters were assessed with a questionnaire for physicians. RESULTS: The direct medical costs totaled 1,667 EUR (range 1,436-1,995 EUR, CI 95%) per patient per 3 months. Charges by physicians were 42 EUR (39-45 EUR, CI 95%) for patients with statutory health insurance and 135 EUR (106-177 EUR, CI 95%) for those with private insurance. Disease severity and disease duration correlated with higher direct medical costs. Motor fluctuations and depression also were major factors influencing cost. CONCLUSION: Our study emphasizes the large economic burden caused mainly by PD medication and hospitalization. For the first time a direct comparison between costs and actual physicians' reimbursement was possible. In combination with further economic studies, this comparison will help to define shortcomings and excesses in PD health care services.

Differences of nine drug resistance interpretation systems in predicting short-term therapy outcomes of treatment-experienced HIV-1 infected patients: a retrospective observational cohort study.

OBJECTIVE: Drug resistance interpretation systems are used to select the optimal antiretroviral therapy in HIV-infected patients. It is unclear how the systems perform in predicting therapy success and failure and in how far the interpretations are affected by insufficient drug levels. METHODS: The accuracy of nine different interpretation systems in predicting therapy outcomes was evaluated using virological, immunological, pharmacological, and clinical data of 130 patients treated at 13 outpatient centers. Individual susceptibility scores of the interpretation systems were converted into active drug scores (ADS) and correlated with therapy success and failure, defined as viral load reduction of equal to or more (n=66) and less than 1 log10 copies/ml (n=64) at three months after drug resistance testing. RESULTS: Three interpretation systems considered the respective therapies as more active compared to the other interpretation systems (p<0.01). These systems predicted therapy success better than the other systems, while the others performed better in predicting therapy failure. Thus, the overall rate of correctly predicted treatment outcomes was comparable between the different systems (73.1-80.0 %). Univariate and multivariate regression analysis revealed significant correlations between the ADS of all interpretation systems and virological therapy outcomes (p<0.0001). In contrast, only three interpretation systems were significantly correlated with immunological therapy outcomes in univariate and just one in multivariate models (p<0.05). Among 128 determinations of drug levels in 64 patient samples, 19.4 % revealed no detectable drug levels. The consideration of insufficient drug levels significantly improved the prediction accuracy of all interpretation systems (p<0.005). CONCLUSION: Differences between interpretation systems in predicting therapy failures and success need to be considered for future consensus algorithms. The prediction accuracy of interpretation systems can be improved by consideration of plasma drug levels.

Online monitoring of BALB/3T3 metabolism and adhesion with multiparametric chip-based system.

A multiparametric chip-based system was employed to measure cell adhesion, metabolism, and response to metal compounds previously classified as cytotoxic in immortalized mouse fibroblasts (BALB/3T3 cell line). The system measures in parallel, online, and in label-free conditions the extracellular acidification rates (with pH-sensitive field effect transistors [ISFETs]), the cellular oxygen consumption (with amperometric electrode structures [Clark-type sensors]), and cell adhesion (with impedimetric interdigitated electrode structures [IDESs]). The experimental protocol was optimized to monitor metabolism and adhesion of the BALB/3T3 cell line. A total of 70,000 cells and a bicarbonate buffer-free running low-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal clone serum III and 1mM Hepes were selected to maintain cells in good conditions on the chip during the measurements performed under perfusion conditions. Cells were exposed to sodium arsenite, cadmium chloride, and cis-platinum at concentrations ranging from 1 to 100 microM. The kinetics of cell response to these compounds was analyzed and suggests that the Clark-type sensors can be more sensitive than IDESs and ISFETs in detecting the presence of high chemical concentration when short exposure times (i.e., 2h) are considered. The cytotoxicity data obtained from the online measurements of acidification, respiration, and adhesion at 24h compare well, in terms of half-inhibition concentration values (IC(50)), with the ones obtained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and colony-forming efficiency (CFE) assay. The results show a good sensitivity of the system combined with the advantages of the online and label-free detection methods that allow following cell status before, during, and after the treatment in the same experiment.

The Faces Symbol Test, a newly developed screening instrument to assess cognitive decline related to multiple sclerosis: first results of the Berlin Multi-Centre FST Validation Study.

Reliable, language-independent, short screening instruments to test for cognitive function in patients with multiple sclerosis (MS) remain rare, despite the high number of patients affected by cognitive decline. We developed a new, short screening instrument, the Faces Symbol Test (FST), and compared its diagnostic test characteristics with a composite of the Digit Symbol Substitution Test (DSST) and the Paced Auditory Serial Addition Test (PASAT), in 108 MS patients and 33 healthy controls. An Informant-Report Questionnaire, a Self-Report Questionnaire, and a neurologist's estimation of the Every Day Life Cognitive Status were also applied to the MS patients. The statistical analyses comprised of a receiver operating characteristic analysis for test accuracy and for confounding variables. The PASAT and DSST composite score estimated that 36.5% of the MS patients had cognitive impairment. The FST estimated that 40.7% of the MS patients were cognitively impaired (sensitivity 84%; specificity 85%). The FST, DSST and PASAT results were significantly correlated with the patients' physical impairment, as measured by the Expanded Disability Status Scale (EDSS). The results suggest that the FST might be a culture-free, sensitive, and practical short screening instrument for the detection of cognitive decline in patients with MS, including those in the early stages.

Simultaneous measurement of cellular respiration and acidification with a single CMOS ISFET.

In vivo, the pH value and oxygen partial pressure are the most important physico-chemical parameters in the microenvironment of human tissues. In vitro, the extracellular acidification rate of cell cultures is an indicator of global cellular metabolism, while the rate of oxygen consumption is a measure of mitochondrial activity. Earlier approaches had the disadvantage that these two values had to be measured with two separate sensors at different loci within the tissue or cell culture. Furthermore, conventional Clark-type oxygen sensors are not very compatible for miniaturisation, making it impossible to measure at small cell volumes or even at the single cell level. We have, therefore, developed an ISFET based sensor structure which is able to measure both pH and oxygen partial pressure. This sensor structure was tested in vitro for simultaneous records of cellular acidification and respiration rates at the same site within the cell culture. This sensor is manufactured by a CMOS-process.

Approach to a multiparametric sensor-chip-based tumor chemosensitivity assay.

Although not widely practiced by oncologists, in vitro tumor chemosensitivity assays (TCA) have proved to increase the lifetime of tumor patients in prospective clinical trials. By individualizing cancer therapy, they can support the clinician's decision which is usually based on empirically retrieved data and thereby prevent inadequate chemotherapy. We present the first results of a new sensor-chip-based technology which might be useful for a multiparametric TCA. In particular, the aspect of dynamic on-line data generation on intact cellular specimens is a major difference to alternative assays. A series of experiments has been performed on cell lines and human tumor explants. Cell cultures and tumor tissue explants were placed on miniaturized silicon and glass sensor chips. The sensor data currently analyze metabolic profiles (rates of extracellular acidification and cellular oxygen consumption) and changes in cell morphology (monitoring of electric impedance). With the cell lines, drug-associated cellular signals have been detected with all three parameters, while primary explants so far caused metabolic responses only. In particular, cellular respiration or mitochondrial activity seems to be a most sensitive indicator of acute cytotoxic effects. The experimental results were achieved using different test versions. Besides giving a status report, the theoretical potential and current problems of sensor chip technology in TCA is discussed.

Multiparametric microsensor chips for screening applications.

The identification of drug targets for pharmaceutical screening can be greatly accelerated by gene databases and expression studies. The identification of leading compounds from growing libraries is realized by high throughput screening platforms. Subsequently, for optimization and validation of identified leading compounds studies of their functionality have to be carried out, and just these functionality tests are a limiting factor. A rigorous preselection of identified compounds by in vitro cellular screening is necessary prior to using the drug candidates for the further time consuming and expensive stage, e.g. in animal models. Our efforts are focused to the parallel development, adaptation and integration of different microelectronic sensors into miniaturized biochips for a multiparametric, functional on-line analysis of living cells in physiologically environments. Parallel and on-line acquisition of data related to different cellular targets is required for advanced stages of drug screening and for economizing animal tests.

Treatment of cells with detergent activates caspases and induces apoptotic cell death.

Due to their amphiphilic properties, detergents readily disrupt cellular membranes and cause rapid cytolysis. In this study we demonstrate that treatment of cells with sublytic concentrations of detergents such as Triton X-100, Nonidet P-40, n-octylglucoside and the bile salt sodium deoxycholate induce typical signs of apoptosis including DNA fragmentation and cleavage of poly(ADP-ribose) polymerase molecules. The detergent concentration required for apoptosis was below the critical micellar concentration. Induction of apoptosis was not restricted to human cells but similarly occurred in a variety of other vertebrate cell lines. Unstimulated peripheral blood mononuclear cells were susceptible to apoptosis induction by detergent suggesting that apoptosis in this circumstance is not mediated by CD95. Cell death was not due to influx of calcium from the medium. Apoptosis was blocked and cytolysis prevented by treatment with peptide inhibitors of caspases. These findings suggest a process of apoptosis that is initiated upon nonspecific alterations at the cell membrane level. Physiologic correlates of this process still have to be defined.

Non-invasive measurement of cell membrane associated proton gradients by ion-sensitive field effect transistor arrays for microphysiological and bioelectronical applications.

The pH in the cellular microenvironment (pH(M)) is an important regulator of cell-to-cell and cell-to-host interactions. Additionally the extracellular acidification rate of a cell culture is an important indicator of global cellular metabolism. In a new approach a biocompatible ion-sensitive field effect transistor (ISFET)-array was developed to measure the pH(M) close to a surface and the global extracellular acidification rate at the same time. This ISFET-array is part of a new multiparametric microsensor chip. The paper highlights some basic applications of this method for in-vitro measurements. Using a fluid perfusion system for cell culture media, it is possible to measure the pH(M) of few (five to ten) adherent tumor cells in a distance of 10-100 nm from the cell plasma membrane. Experiments showed a pH(M)-value of 6.68 +/- 0.06 pH. Further experiments suggest that both the low pH, and the extracellular acidification rate of the examined tumor cell line are mainly built up by glycolysis.

On-line control of cellular adhesion with impedance measurements using interdigitated electrode structures.

Critical parameters to be assessed in cell culture are the number of viable cells and cell viability. Growth, product formation, toxicity effects and the overall success of cell culture can depend largely on these. With interdigitated electrode structures (IDES) adherent cells are cultured directly on a pair of interdigitated electrodes, and the impedance of the system gives insight into the adhesive behaviour of the cells. The signal is influenced by the changes in number, growth and morphological behaviour of adherently growing cells, mainly owing to the insulating effects of the cell membranes. Five different cell lines are used, and their divergent behaviour is monitored over a period of four days, from inoculation of the cells to killing of the cells at the end of the experiments. Even when the cells from close monolayers, great fluctuations in the impedance signal can be observed. Nevertheless, for a more complete description of cellular systems, other parameters, such as acidification and respiration, have to be included in the measuring system.

Microsensor-aided measurements of cellular signalling and metabolism on tumor cells. The cell monitoring system (cms(R)).

Microsensors provide instruments particularly suited for the rapid, noninvasive and on-line analysis of cell and tissue cultures. The microsensor system presented in this paper is a modular arrangement of various planar and nonplanar sensor elements for the measurement of physiological parameters of cell cultures. An optic access to the cultures (e.g. for light microscopy and spectrophotometric techniques) is also provided for a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy (predicative chemotherapy assays) and pharmacology but it turned out to be also an effective tool for toxicological and environmental research.

Monitoring of cellular signalling and metabolism with modular sensor-technique: the PhysioControl-Microsystem (PCM).

Microsensors provide instruments particularly suited for the noninvasive analysis of cell and tissue cultures. The outstanding benefit lies in the passive behaviour of continuously working transducers, which in turn allows the dynamic recording of function-specific cellular processes. The microsensor system presented in this paper is a modular arrangement of various planar and non-planar sensor elements surrounding small cell culture chambers. An optic access to the cultures (e.g. for high resolution light microscopy and spectro-photometric techniques) enables a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy and pharmacology but it proved to be an effective device both for toxicological and environmental research.

Isolated extracellular matrix-based three-dimensional in vitro models to study orthotopically cancer cell infiltration and invasion.

An initial event in colon cancer progression is the migration of epithelial cells through the basement membrane (BM) and the invasion of the colon submucosa, where tumour cells enter blood and lymph vessels to spread throughout the body. To interrupt this process would mean the prevention of metastasis. In order to investigate tumour cell invasion orthotopically in the human system, we established novel in vitro models which mimic normal human colon tissue (colon reproductions, CoRes) and primary colon carcinomas (artificial tumours, ArTs). These models are based on the isolated extracellular matrix (iECM) of the respective human tissues. Two isolation methods were established, the Digestion Method and the Lysis Method neither of which destroyed the characteristic architecture of the ECM found in the original tissues. BM components, i.e. laminin, fibronectin and collagen IV, were detectable in the iECM isolated with the Lysis Method but not those isolated with the Digestion Method. Scanning electron microscopic analysis of the normal colon iECM demonstrated that even if the BM was missing, the luminal surface consisted of densely packed ECM filaments which do not allow cell infiltration without degradation of the iECM. Furthermore, we demonstrated that iECM can be separately supplemented with different cell types, i.e. colorectal carcinoma cells, normal fibroblasts and immune cells at any desired concentration, combination and localisation. Therefore, these models could be used to determine the role of the BM and of the tumour cell/normal cell crosstalk in the infiltration process of human colorectal carcinoma cells.

Human immunodeficiency virus glycoprotein-specific CD4+ cytotoxic T lymphocytes are involved in two types of cytotoxicity: antigen-specific and cell-cell fusion-related cell lysis.

Human immunodeficiency virus (HIV) glycoprotein-specific CD4+ cytotoxic T lymphocytes (CTLs) lyse target cells in an MHC-restricted calcium-dependent fashion similar to the mechanism used by CD8+ CTLs. However, contact of unprimed peripheral blood CD4+ T cells with HIV glycoprotein-expressing cells has been shown to cause, in addition to cell-cell fusion, rapid cytolysis that may resemble antigen-specific cytotoxicity in the chromium release assay. In this study, the ability of glycoprotein-specific CD4+ CTLs to undergo similar fusion-related cytolysis was examined. The data obtained demonstrate that in addition to antigen-specific calcium-dependent cytotoxicity, envelope-specific CD4+ CTLs are involved in fusion-related, calcium-independent cytolysis.

Vaccinia virus serpin B13R (SPI-2) inhibits interleukin-1beta-converting enzyme and protects virus-infected cells from TNF- and Fas-mediated apoptosis, but does not prevent IL-1beta-induced fever.

The vaccinia virus (VV) strain Western Reserve B13R gene encodes a 38.5 kDa intracellular polypeptide that is non-essential for virus replication in vitro and does not affect virus virulence in a murine intranasal model. The protein has 92% amino acid identity with the cowpox virus cytokine response modifier A (crmA) protein which inhibits the interleukin (IL)-1beta converting enzyme (ICE). Here, we show that extracts from THP-1 cells infected with VV strains expressing B13R prevent the cleavage of in vitro transcribed and translated pro-IL-1beta into mature IL-1beta. Similarly, THP-1 cells infected with VVs expressing B13R process pro-IL-1beta into mature IL-1beta inefficiently in situ. Despite its inhibition of ICE, B13R does not prevent fever in infected mice, a systemic effect mediated by IL-1beta. Instead, fever is controlled by the VV IL-1beta receptor, encoded by gene B15R, and deletion of both the B13R and B15R genes did not increase the febrile response compared to deletion of B15R alone. The B13R protein does, however, block apoptosis mediated by anti-Fas antibodies or by tumour necrosis factor (TNF) and cycloheximide. Using DNA fragmentation, chromium release and microscopic analyses it was shown that cells infected with wild-type VV strain WR, or a revertant virus in which the B13R gene had been re-inserted into the B13R deletion mutant, are more resistant than uninfected cells or deletion mutant-infected cells to apoptosis mediated by anti-Fas and TNF.

Monitoring of cellular behaviour by impedance measurements on interdigitated electrode structures.

A new method for on-line and real-time monitoring of concentration, growth and physiological state of cells in culture is described. This biosensor is based on impedance measurements of adherently growing cells on interdigitated electrode structures (IDES). The measurements can be performed for several days as there is no detectable electrical influence on the cells. The versatility of this new sensor is shown with some exemplary experiments. Cell density, growth and long-term behaviour of cells on the electrodes clearly change the impedance of the IDES. Both, the global influence of serum components (deprivation of foetal bovine serum) and the toxic effects of heavy metal ions (cadmium) result in changes of the sensor signal and can be visualized.