Spiez Laboratory

Spiez, Switzerland

Spiez Laboratory

Spiez, Switzerland
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Comim C.M.,Health Science University | Barichello T.,Health Science University | Grandgirard D.,University of Bern | Grandgirard D.,Spiez Laboratory | And 4 more authors.
Molecular Neurobiology | Year: 2013

The brain is one of the first organs affected during sepsis development resulting in apoptosis for a short-term and cognitive impairment for a long-term. Despite its importance, the mechanisms of brain dysfunction during sepsis are not fully elucidated. Thus, we here, in an animal model of sepsis, evaluated apoptosis in the dentate gyrus cell layer of the hippocampus to document the involvement of caspase-3 in the pathogenesis of neuronal apoptosis. Wistar rats sham-operated or submitted to the cecal ligation and perforation (CLP) procedure were killed at 12, 24, 48 h, and 10 days after surgery for the determination of caspase-3 and apoptosis rate. In a separate cohort of animals, a caspase-3-specific inhibitor was administered and animals were killed at 12 h after sepsis. An increase in the number of apoptotic cells 12, 24, and 48 h by histopathological evaluations and an increase of caspase-3 apoptotic cells 12 and 24 h after sepsis induction were observed. The caspase-3 inhibitor decreases the number of apoptotic cells by histopathological evaluations but not by immunohistochemistry evaluations. Caspase-3 is involved in part in apoptosis in the dentate gyrus cell layer of the hippocampus in septic rats submitted by CLP. © 2012 Springer Science+Business Media New York.


Grandgirard D.,University of Bern | Burri M.,University of Bern | Agyeman P.,University of Bern | Leib S.L.,University of Bern | Leib S.L.,Spiez Laboratory
Antimicrobial Agents and Chemotherapy | Year: 2012

Exacerbation of cerebrospinal fluid (CSF) inflammation in response to bacteriolysis by beta-lactam antibiotics contributes to brain damage and neurological sequelae in bacterial meningitis. Daptomycin, a nonlytic antibiotic acting on Gram-positive bacteria, lessens inflammation and brain injury compared to ceftriaxone. With a view to a clinical application for pediatric bacterial meningitis, we investigated the effect of combining daptomycin or rifampin with ceftriaxone in an infant rat pneumococcal meningitis model. Eleven-day-old Wistar rats with pneumococcal meningitis were randomized to treatment starting at 18 h after infection with (i) ceftriaxone (100 mg/kg of body weight, subcutaneously [s.c.], twice a day [b.i.d.]), (ii) daptomycin (10 mg/kg, s.c., daily) followed 15 min later by ceftriaxone, or (iii) rifampin (20 mg/kg, intraperitoneally [i.p.], b.i.d.) followed 15 min later by ceftriaxone. CSF was sampled at 6 and 22 h after the initiation of therapy and was assessed for concentrations of defined chemokines and cytokines. Brain damage was quantified by histomorphometry at 40 h after infection and hearing loss was assessed at 3 weeks after infection. Daptomycin plus ceftriaxone versus ceftriaxone significantly (P<0.04) lowered CSF concentrations of monocyte chemoattractant protein 1 (MCP-1), MIP-1α, and interleukin 6 (IL-6) at 6 h and MIP-1α, IL-6, and IL-10 at 22 h after initiation of therapy, led to significantly (P<0.01) less apoptosis, and significantly (P<0.01) improved hearing capacity. While rifampin plus ceftriaxone versus ceftriaxone also led to lower CSF inflammation (P<0.02 for IL-6 at 6 h), it had no significant effect on apoptosis and hearing capacity. Adjuvant daptomycin could therefore offer added benefits for the treatment of pediatric pneumococcal meningitis. Copyright © 2012, American Society for Microbiology. All Rights Reserved.


Konig S.,University of Bern | Aebi B.,Spiez Laboratory | Lanz S.,University of Bern | Gasser M.,University of Bern | Weinmann W.,University of Bern
Analytical and Bioanalytical Chemistry | Year: 2011

A universal and robust analytical method for the determination of Δ9-tetrahydrocannabinol (THC) and two of its metabolites Δ9-(11-OH)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-carboxy- tetrahydrocannabinol (THC-COOH) in human whole blood was developed and validated for use in forensic toxicology. Protein precipitation, integrated solid phase extraction and on-line enrichment followed by high-performance liquid chromatography separation and detection with a triple quadrupole mass spectrometer were combined. The linear ranges used for the three cannabinoids were from 0.5 to 20 ng/mL for THC and 11-OH-THC and from 2.5 to 100 ng/mL for THC-COOH, therefore covering the requirements for forensic use. Correlation coefficients of 0.9980 or better were achieved for all three analytes. No relevant hydrolysis was observed for THC-COOH glucuronide with this procedure - in contrast to our previous GC-MS procedure, which obviously lead to an artificial increase of the THC-COOH concentration due to the hydrolysis of the glucuronide-conjugate occurring at high pH during the phase-transfer catalyzed methylation step. © 2011 Springer-Verlag.


Wittwer M.,Spiez Laboratory | Heim J.,Spiez Laboratory | Schar M.,Spiez Laboratory | Dewarrat G.,Spiez Laboratory | Schurch N.,Spiez Laboratory
Systematic and Applied Microbiology | Year: 2011

In the everyday routine of an analytic lab, one is often confronted with the challenge to identify an unknown microbial sample lacking prior information to set the search limits.In the present work, we propose a workflow, which uses the spectral diversity of a commercial database (SARAMIS) to narrow down the search field at a certain taxonomic level, followed by a refined classification by supervised modelling. As supervised learning algorithm, we have chosen a shrinkage discriminant analysis approach, which takes collinearity of the data into account and provides a scoring system for biomarker ranking. This ranking can be used to tailor specific biomarker subsets, which optimize discrimination between subgroups, allowing a weighting of misclassification.The suitability of the approach was verified based on a dataset containing the mass spectra of three Yersinia species Yersinia enterocolitica, Y. pseudotuberculosis and Yersinia pestis. Thereby, we laid the emphasis on the discrimination between the highly related species Yersinia pseudotuberculosis and Y. pestis.All three species were correctly identified at the genus level by the commercial database. Whereas Y. enterocolitica was correctly identified at the species level, discrimination between the highly related Y. pseudotuberculosis and Y. pestis strains was ambiguous. With the use of the supervised modelling approach, we were able to accurately discriminate all the species even when grown under different culture conditions. © 2010 Elsevier GmbH.


Gaumann R.,University of Bern | Muhlemann K.,University of Bern | Strasser M.,Spiez Laboratory | Beuret C.M.,Spiez Laboratory
Applied and Environmental Microbiology | Year: 2010

Tick-borne encephalitis (TBE), a viral infection of the central nervous system, is endemic in many Eurasian countries. In Switzerland, TBE risk areas have been characterized by geographic mapping of clinical cases. Since mass vaccination should significantly decrease the number of TBE cases, alternative methods for exposure risk assessment are required. We established a new PCR-based test for the detection of TBE virus (TBEV) in ticks. The protocol involves an automated, high-throughput nucleic acid extraction method (QIAsymphony SP system) and a one-step duplex real-time reverse transcription-PCR (RT-PCR) assay for the detection of European subtype TBEV, Including an internal process control. High usability, reproducibility, and equivalent performance for virus concentrations down to 5 × 103 viral genome equivalents/μl favor the automated protocol compared to the modified guanidinium thiocyanate-phenol-chloroform extraction procedure. The real-time RT-PCR allows fast, sensitive (limit of detection, 10 RNA copies/μl), and specific (no false-positive test results for other TBEV subtypes, other fiaviviruses, or other tick-transmitted pathogens) detection of European subtype TBEV. The new detection method was applied in a national surveillance study, in which 62,343 Ixodes ricinus ticks were screened for the presence of TBE virus. A total of 38 foci of endemicity could be identified, with a mean virus prevalence of 0.46%. The foci do not fully agree with those defined by disease mapping. Therefore, the proposed molecular test procedure constitutes a prerequisite for an appropriate TBE surveillance. Our data are a unique complement of human TBE disease case mapping in Switzerland. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


A novel active capillary dielectric barrier discharge plasma ionization (DBDI) technique for mass spectrometry is applied to the direct detection of 13 chemical warfare related compounds, including sarin, and compared to secondary electrospray ionization (SESI) in terms of selectivity and sensitivity. The investigated compounds include an intact chemical warfare agent and structurally related molecules, hydrolysis products and/or precursors of highly toxic nerve agents (G-series, V-series, and "new" nerve agents), and blistering and incapacitating warfare agents. Well-defined analyte gas phase concentrations were generated by a pressure-assisted nanospray with consecutive thermal evaporation and dilution. Identification was achieved by selected reaction monitoring (SRM). The most abundant fragment ion intensity of each compound was used for quantification. For DBDI and SESI, absolute gas phase detection limits in the low ppt range (in MS/MS mode) were achieved for all compounds investigated. Although the sensitivity of both methods was comparable, the active capillary DBDI sensitivity was found to be dependent on the applied AC voltage, thus enabling direct tuning of the sensitivity and the in-source fragmentation, which may become a key feature in terms of field applicability. Our findings underline the applicability of DBDI and SESI for the direct, sensitive detection and quantification of several CWA types and their degradation products. Furthermore, they suggest the use of DBDI in combination with hand-held instruments for CWAs on-site monitoring. © 2014 American Chemical Society.


Liechti F.D.,University of Bern | Grandgirard D.,University of Bern | Leppert D.,Hoffmann-La Roche | Leib S.L.,University of Bern | Leib S.L.,Spiez Laboratory
Infection and Immunity | Year: 2014

Pneumococcal meningitis (PM) results in high mortality rates and long-lasting neurological deficits. Hippocampal apoptosis and cortical necrosis are histopathological correlates of neurofunctional sequelae in rodent models and are frequently observed in autopsy studies of patients who die of PM. In experimental PM, inhibition of matrix metalloproteinases (MMPs) and/or tumor necrosis factor (TNF)-converting enzyme (TACE) has been shown to reduce brain injury and the associated impairment of neurocognitive function. However, none of the compounds evaluated in these studies entered clinical development. Here, we evaluated two second-generation MMP and TACE inhibitors with higher selectivity and improved oral availability. Ro 32-3555 (Trocade, cipemastat) preferentially inhibits collagenases (MMP-1, -8, and -13) and gelatinase B (MMP-9), while Ro 32-7315 is an efficient inhibitor of TACE. PM was induced in infant rats by the intracisternal injection of live Streptococcus pneumoniae. Ro 32-3555 and Ro 32-7315 were injected intraperitoneally, starting at 3 h postinfection. Antibiotic (ceftriaxone) therapy was initiated at 18 h postinfection, and clinical parameters (weight, clinical score, mortality rate) were recorded. Myeloperoxidase activities, concentrations of cytokines and chemokines, concentrations of MMP-2 and MMP-9, and collagen concentrations were measured in the cerebrospinal fluid. Animals were sacrificed at 42 h postinfection, and their brains were assessed by histomorphometry for hippocampal apoptosis and cortical necrosis. Both compounds, while exhibiting disparate MMP and TACE inhibitory profiles, decreased hippocampal apoptosis and cortical injury. Ro 32-3555 reduced mortality rates and cerebrospinal fluid TNF, interleukin-1β (IL-1β) and collagen levels, while Ro 32-7315 reduced weight loss and cerebrospinal fluid TNF and IL-6 levels. © 2014, American Society for Microbiology.


An LC-UV-SPE NMR method for the analysis of polar hydrolysis products of the chemical warfare agents known as sulfur mustards at low ppm levels in environmental samples is developed. The hydrolysis products thiodiglycol (I), bis(2-hydroxyethylthio)methane (II), 1,2-bis(2-hydroxyethylthio)ethane (III), 1,3-bis(2-hydroxyethylthio)propane (IV), 1,4-bis(2-hydroxyethylthio)butane (V), 1,5-bis(2-hydroxyethylthio)pentane (VI), bis(2-hydroxymethylthioethyl)ether (VII) and bis(2-hydroxyethylthioethyl)ether (VIII) are baseline separated within 11min by the LC gradient program and trapped post-column on SPE cartridges. After elution in 2mm o.d. NMR tubes 1H NMR spectra were recorded. Recoveries vary from 43±5 for I to 102±5% for VI and are limited by volume breakthrough. The detection limits of the LC-SPE NMR method vary between 200ng for V and 450ng for I. Increasing the injection volume is shown to be more effective than multiple trapping for the analytes I-VIII to increase the amount of material trapped on the SPE cartridges. The applicability of the developed method to the analysis of environmental samples was tested by the analysis of sample 293 provided by the 29th Official OPCW (Organization for the Prohibition of Chemical Weapons) Proficiency Test. The chromatographic and 1H NMR data obtained by the method are highly reproducible and provide acceptable data for the identification of chemicals related to CWC (Chemical Weapons Convention) in case an off-site analysis for the verification of the CWC or OPCW Proficiency Tests according to the OPCW criteria for the acceptance of chromatographic and 1H NMR spectral data. © 2013 Elsevier B.V.


Blaser C.,University of Bern | Wittwer M.,Spiez Laboratory | Grandgirard D.,University of Bern | Leib S.L.,University of Bern
PLoS ONE | Year: 2011

Streptococcus pneumoniae is the most common pathogen causing non-epidemic bacterial meningitis worldwide. The immune response and inflammatory processes contribute to the pathophysiology. Hence, the anti-inflammatory dexamethasone is advocated as adjuvant treatment although its clinical efficacy remains a question at issue. In experimental models of pneumococcal meningitis, dexamethasone increased neuronal damage in the dentate gyrus. Here, we investigated expressional changes in the hippocampus and cortex at 72 h after infection when dexamethasone was given to infant rats with pneumococcal meningitis. Nursing Wistar rats were intracisternally infected with Streptococcus pneumoniae to induce experimental meningitis or were sham-infected with pyrogen-free saline. Besides antibiotics, animals were either treated with dexamethasone or saline. Expressional changes were assessed by the use of GeneChip® Rat Exon 1.0 ST Arrays and quantitative real-time PCR. Protein levels of brain-derived neurotrophic factor, cytokines and chemokines were evaluated in immunoassays using Luminex xMAP® technology. In infected animals, 213 and 264 genes were significantly regulated by dexamethasone in the hippocampus and cortex respectively. Separately for the cortex and the hippocampus, Gene Ontology analysis identified clusters of biological processes which were assigned to the predefined categories "inflammation", "growth", "apoptosis" and others. Dexamethasone affected the expression of genes and protein levels of chemokines reflecting diminished activation of microglia. Dexamethasone-induced changes of genes related to apoptosis suggest the downregulation of the Akt-survival pathway and the induction of caspase-independent apoptosis. Signalling of pro-neurogenic pathways such as transforming growth factor pathway was reduced by dexamethasone resulting in a lack of pro-survival triggers. The anti-inflammatory properties of dexamethasone were observed on gene and protein level in experimental pneumococcal meningitis. Further dexamethasone-induced expressional changes reflect an increase of pro-apoptotic signals and a decrease of pro-neurogenic processes. The findings may help to identify potential mechanisms leading to apoptosis by dexamethasone in experimental pneumococcal meningitis. © 2011 Blaser, et al.


Liechti F.D.,University of Bern | Grandgirard D.,University of Bern | Leib S.L.,University of Bern | Leib S.L.,Spiez Laboratory
Neuroscience | Year: 2015

Background: High mortality and morbidity rates are observed in patients with bacterial meningitis (BM) and urge for new adjuvant treatments in addition to standard antibiotic therapies. In BM the hippocampal dentate gyrus is injured by apoptosis while in cortical areas ischemic necrosis occurs. Experimental therapies aimed at reducing the inflammatory response and brain damage have successfully been evaluated in animal models of BM. Fluoxetine (FLX) is an anti-depressant of the selective serotonin reuptake inhibitors (SSRI) and was previously shown to be neuroprotective in vitro and in vivo. We therefore assessed the neuroprotective effect of FLX in experimental pneumococcal meningitis. Methods: Infant rats were infected intracisternally with live Streptococcus pneumoniae. Intraperitoneal treatment with FLX (10mgkg-1d-1) or an equal volume of NaCl was initiated 15min later. 18, 27, and 42h after infection, the animals were clinically (weight, clinical score, mortality) evaluated and subject to a cisternal puncture and inflammatory parameters (i.e., cyto-/chemokines, myeloperoxidase activity, matrix metalloproteinase concentrations) were measured in cerebrospinal fluid (CSF) samples. At 42h after infection, animals were sacrificed and the brains collected for histomorphometrical analysis of brain damage. Results: A significant lower number of animals treated with FLX showed relevant hippocampal apoptosis when compared to littermates (9/19 animals vs 18/23, P=. 0.038). A trend for less damage in cortical areas was observed in FLX-treated animals compared to controls (13/19 vs 13/23, P=. ns). Clinical and inflammatory parameters were not affected by FLX treatment. Conclusion: A significant neuroprotective effect of FLX on the hippocampus was observed in acute pneumococcal meningitis in infant rats. © 2015 IBRO.

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