Primary bovine synoviocyte cultures: a useful tool for in vitro drug testing?

The aim of the study was to evaluate bovine synoviocyte culture as an in vitro model to test new intra-articular drugs. The inflammatory reaction pattern of synoviocytes as compared to fibroblasts was studied over nine passages. Expression of pro-inflammatory cytokines was assessed after stimulation with lipopolysaccharide. Immunohistochemical markers were used to identify synoviocyte populations. Primary synoviocytes expressed markedly higher amounts of interleukin-1beta mRNA and tumour necrosis factor-alpha mRNA than fibroblasts after stimulation. This difference was lost over two passages. CD68-positive macrophage-like synoviocytes diminished over three passages, which may explain the reduced pro-inflammatory cytokine response. Primary bovine synoviocytes appear to be an appropriate and optimised model for testing novel drugs for cattle, because their response may more closely reflect in vivo tissue responses compared to cultured cell lines.

Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP LC-MS/MS system and library searching

The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a "scheduled" survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization-MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography-mass spectrometry, and liquid chromatography-diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).

In vitro differentiation of near-unlimited numbers of functional mouse basophils using conditional Hoxb8

Background: Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. Methods: A method is described to massively expand bone marrow–derived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. Results: IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). Conclusion: A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.

Rapid and simple LC-MS/MS-Screening of 64 novel psychoactive substances using dried blood spots

The range of novel psychoactive substances (NPS) including phenethylamines, cathinones, piperazines, tryptamines, etc. is continuously growing. Therefore, fast and reliable screening methods for these compounds are essential and needed. The use of dried blood spots (DBS) for a fast straightforward approach helps to simplify and shorten sample preparation significantly. DBS were produced from 10 µl of whole blood and extracted offline with 500 µl methanol followed by evaporation and reconstitution in mobile phase. Reversed-phase chromatographic separation and mass spectrometric detection (RP-LC-MS/MS) was achieved within a run time of 10 min. The screening method was validated by evaluating the following parameters: limit of detection (LOD), matrix effect, selectivity and specificity, extraction efficiency, and short-term and long-term stability. Furthermore, the method was applied to authentic samples and results were compared with those obtained with a validated whole blood method used for Routine analysis of NPS. LOD was between 1 and 10 ng/ml. No interference from Matrix compounds was observed. The method was proven to be specific and selective for the analytes, although with limitations for 3-FMC/flephedrone and MDDMA/MDEA. Mean extraction efficiency was 84.6 %. All substances were stable in DBS for at least a week when cooled. Cooling was essential for the stability of cathinones. Prepared samples were stable for at least 3 days. Comparison to the validated whole blood method yielded similar results. DBS were shown to be useful in developing a rapid screening method for NPS with simplified sample preparation. Copyright © 2013 John Wiley & Sons, Ltd

Pharmacokinetics of GHB and detection window in Serum and Urine after single uptake of a low dose of GLB - an Experiment with two volunteers

During the last few years γ-hydroxybutyric acid (GHB) and γ-butyrolactone (GBL) have attracted much interest as recreational drugs and knock-out drops in drug-facilitated sexual assaults. This experiment aims at getting an insight into the pharmacokinetics of GHB after intake of GBL. Therefore Two volunteers took a single dose of 1.5 ml GBL, which had been spiked to a soft drink. Assuming that GBL was completely metabolized to GHB, the corresponding amount of GHB was 2.1 g. Blood and urine samples were collected 5 h and 24 h after ingestion, respectively. Additionally, hair samples (head hair and beard hair) were taken within four to five weeks after intake of GBL. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after protein precipitation with acetonitrile. The following observations were made: spiked to a soft drink, GBL, which tastes very bitter, formed a liquid layer at the bottom of the glass, only disappearing when stirring. Both volunteers reported weak central effects after approximately 15 min, which disappeared completely half an hour later. Maximum concentrations of GHB in serum were measured after 20 min (95 µg/ml and 106 µg/ml). Already after 4-5 h the GHB concentrations in serum decreased below 1 µg/ml. In urine maximum GHB concentrations (140 µg/ml and 120 µg/ml) were measured after 1-2 h, and decreased to less than 1 µg/ml within 8-10 h. The Ratio of GHB in serum versus blood was 1.2 and 1.6

Generation of a murine hepatic angiosarcoma cell line and reproducible mouse tumor model

Hepatic angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited as animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aims of this study were to: (1) establish and characterize a cell line derived from this murine AS, (2) identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and (3) generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and Von Willebrand factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors, confirming the tumorigenic potential of these cells. In summary, we established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, supporting further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing.Laboratory Investigation advance online publication, 24 November 2014; doi:10.1038/labinvest.2014.141.

Quantification of anandamide and 2-arachidonoylglycerol plasma levels to examine potential influences of tetrahydrocannabinol application on the endocannabinoid system in humans

The effects of tetrahydrocannabinol (THC) and endogenous cannabinoids (endocannabinoids, ECs) are both mediated by activation of the cannabinoid receptors CB1 and CB2. Exogenous activation of these receptors by THC could therefore alter EC levels. We tested this hypothesis in healthy volunteers (n = 25) who received a large intravenous dose of THC (0.10 mg/kg). Effects on the EC system were quantified by serial measurements of plasma ECs after THC administration. Eleven blood samples were drawn during the first 5 h after THC administration and two more samples after 24 and 48 h. THC, its metabolites THC-OH (biologically active) and THC-COOH (non-active), and the ECs anandamide and 2-arachidonoylglycerol (2-AG) were quantified by liquid chromatography-mass spectrometry. EC-plasma levels showed a biphasic response after THC injection reaching maximal values at 30 min. Anandamide increased slightly from 0.58 ± 0.21 ng/ml at baseline to 0.64 ± 0.24 ng/ml (p < 0.05) and 2-AG from 7.60 ± 4.30 ng/ml to 9.50 ± 5.90 ng/ml (p < 0.05). After reaching maximal concentrations, EC plasma levels decreased markedly to a nadir of 300 min after THC administration (to 0.32 ± 0.15 ng/ml for anandamide and to 5.50 ± 3.01 ng/ml for 2-AG, p < 0.05). EC plasma concentrations returned to near baseline levels until 48 h after the experiment. THC (0.76 ± 0.16 ng/ml) and THC-OH (0.36 ± 0.17 ng/ml) were still measurable at 24 h and remained detectible until 48 h after THC administration. Although the underlying mechanism is not clear, high doses of intravenous THC appear to influence endogenous cannabinoid concentrations and presumably EC-signalling.

Stable expression of Escherichia coli beta-glucuronidase A (GusA) in Giardia lamblia: application to high-throughput drug susceptibility testing

OBJECTIVES: In order to create a suitable model for high-throughput drug screening, a Giardia lamblia WB C6 strain expressing Escherichia coli glucuronidase A (GusA) was created and tested with respect to susceptibility to the anti-giardial drugs nitazoxanide and metronidazole. METHODS: GusA, a well-established reporter gene in other systems, was cloned into the vector pPacVInteg allowing stable expression in G. lamblia under control of the promoter from the glutamate dehydrogenase (gdh) gene. The resulting transgenic strain was compared with the wild-type strain in a vitality assay, characterized with respect to susceptibility to nitazoxanide, metronidazole and -- as assessed in a 96-well plate format -- to a panel of 15 other compounds to be tested for anti-giardial activity. RESULTS: GusA was stably expressed in G. lamblia. Using a simple glucuronidase assay protocol, drug efficacy tests yielded results similar to those from cell counting. CONCLUSIONS: G. lamblia WB C6 GusA is a suitable tool for high-throughput anti-giardial drug screening.

Preclinical testing of strategies for therapeutic targeting of human T-cell trafficking in vivo

Naive T cells are migratory cells that continuously recirculate between blood and lymphoid tissues. Antigen-specific stimulation of T cells within the lymph nodes reprograms the trafficking properties of T cells by inducing a specific set of adhesion molecules and chemokine receptors on their surface which allow these activated and effector T cells to effectively and specifically home to extralymphoid organs. The observations of organ-specific homing of T cells initiated the development of therapeutic strategies targeting adhesion receptors for organ-specific inhibition of chronic inflammation. As most adhesion receptors have additional immune functions besides mediating leukocyte trafficking, these drugs may have additional immunomodulatory effects. Therapeutic targeting of T-cell trafficking to the central nervous system is the underlying concept of a novel treatment of relapsing remitting multiple sclerosis with the humanized anti-alpha-4-integrin antibody natalizumab. In this chapter, we describe a possible preclinical in vivo approach to directly visualize the therapeutic efficacy of a given drug in inhibiting T-cell homing to a certain organ at the example of the potential of natalizumab to inhibit the trafficking of human T cells to the inflamed central nervous system in an animal model of multiple sclerosis.

Replicative phenotyping adds value to genotypic resistance testing in heavily pre-treated HIV-infected individuals--the Swiss HIV Cohort Study

Background Replicative phenotypic HIV resistance testing (rPRT) uses recombinant infectious virus to measure viral replication in the presence of antiretroviral drugs. Due to its high sensitivity of detection of viral minorities and its dissecting power for complex viral resistance patterns and mixed virus populations rPRT might help to improve HIV resistance diagnostics, particularly for patients with multiple drug failures. The aim was to investigate whether the addition of rPRT to genotypic resistance testing (GRT) compared to GRT alone is beneficial for obtaining a virological response in heavily pre-treated HIV-infected patients. Methods Patients with resistance tests between 2002 and 2006 were followed within the Swiss HIV Cohort Study (SHCS). We assessed patients' virological success after their antiretroviral therapy was switched following resistance testing. Multilevel logistic regression models with SHCS centre as a random effect were used to investigate the association between the type of resistance test and virological response (HIV-1 RNA <50 copies/mL or ≥1.5log reduction). Results Of 1158 individuals with resistance tests 221 with GRT+rPRT and 937 with GRT were eligible for analysis. Overall virological response rates were 85.1% for GRT+rPRT and 81.4% for GRT. In the subgroup of patients with >2 previous failures, the odds ratio (OR) for virological response of GRT+rPRT compared to GRT was 1.45 (95% CI 1.00-2.09). Multivariate analyses indicate a significant improvement with GRT+rPRT compared to GRT alone (OR 1.68, 95% CI 1.31-2.15). Conclusions In heavily pre-treated patients rPRT-based resistance information adds benefit, contributing to a higher rate of treatment success.

Oral prednisolone induced acute generalized exanthematous pustulosis due to corticosteroids of group A confirmed by epicutaneous testing and lymphocyte transformation tests

BACKGROUND: Acute generalized exanthematous pustulosis (AGEP) is a rare cutaneous eruption which is often provoked by drugs. CASE REPORT: We report 2 cases of AGEP which showed rapidly spreading pustular eruptions accompanied by malaise, fever and neutrophilia after the administration of systemic prednisolone (corticosteroid of group A, hydrocortisone type). The histological examination showing neutrophilic subcorneal spongiform pustules was consistent with the diagnosis of AGEP. In both cases the rash cleared within a week upon treatment with topical steroids (corticosteroid of group D1, betamethasonedipropionate type and corticosteroid of group D2, hydrocortisone-17-butyrate type). Three months after recovery, the sensitization to corticosteroids of group A was confirmed by epicutaneous testing and positive lymphocyte transformation tests. CONCLUSION: These cases show that systemic corticosteroids can induce AGEP and demonstrate that epicutaneous testing and lymphocyte transformation tests may be helpful in identifying the causative drug. Our data support previous reports indicating an important role for drug-specific T cells in inducing neutrophil inflammation in this disease.

Multidrug- and extensively drug-resistant tuberculosis

We evaluated risk factors and treatment outcomes associated with multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) in Germany in 2004–2006. In 177 (4%) of 4,557 culture-positive TB cases, Mycobacterium tuberculosis isolates were identified as MDR TB; an additional 7 (0.15%) met criteria for XDR TB. Of these 184 patients, 148 (80%) were born in countries of the former Soviet Union. In patients with XDR TB, hospitalization was longer (mean ± SD 202 ± 130 vs. 123 ± 81 days; p = 0.015) and resistance to all first-line drugs was more frequent (36% vs. 86%; p = 0.013) than in patients with MDR TB. Seventy-four (40%) of these 184 patients received treatment with linezolid. Treatment success rates ranged from 59% for the entire cohort (59% for MDR TB and 57% for XDR TB) to 87% for those with a definitive outcome (n = 125; 89% for MDR TB and 80% for XDR TB). Extensive drug susceptibility testing and availability of second- and third-line drugs under inpatient management conditions permit relatively high treatment success rates in MDR- and XDR TB.