A European multicenter study of immunoblotting in serodiagnosis of lyme borreliosis

A European multicenter study of immunoblotting for the serodiagnosis of Lyme borreliosis showed considerable variation in results obtained from tests with a panel of 227 serum samples. Six laboratories used different immunoblot methods, and a wide range of bands was detected in all the assays. Multivariable logistic regression analysis of data from individual laboratories was used to determine the most discriminatory bands for reliable detection of antibodies to Borrelia burgdorferi sensu lato. These bands were used to construct individual interpretation rules for the immunoblots used in the six laboratories. Further analysis identified a subset of eight bands, which were important in all the laboratories, although with variations in significance. Possible European rules, all closely related, were formulated from these bands, although there was no single rule that gave high levels of sensitivity and specificity for all the laboratories. This is a reflection of the wide range of methodologies used, especially the use of different species and strains of B. burgdorferi sensu lato. The panel of European rules provides a framework for immunoblot interpretation which may be adapted in relation to the characteristics of Lyme borreliosis in local areas.     

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines

Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes.     

Assessing the spatiotemporal evolution of neuronal activation with single-trial event-related potentials and functional MRI

The brain acts as an integrated information processing system, which methods in cognitive neuroscience have so far depicted in a fragmented fashion. Here, we propose a simple and robust way to integrate functional MRI (fMRI) with single trial event-related potentials (ERP) to provide a more complete spatiotemporal characterization of evoked responses in the human brain. The idea behind the approach is to find brain regions whose fMRI responses can be predicted by paradigm-induced amplitude modulations of simultaneously acquired single trial ERPs. The method was used to study a variant of a two-stimulus auditory target detection (odd-ball) paradigm that manipulated predictability through alternations of stimulus sequences with random or regular target-to-target intervals. In addition to electrophysiologic and hemodynamic evoked responses to auditory targets per se, single-trial modulations were expressed during the latencies of the P2 (170-ms), N2 (200-ms), and P3 (320-ms) components and predicted spatially separated fMRI activation patterns. These spatiotemporal matches, i.e., the prediction of hemodynamic activation by time-variant information from single trial ERPs, permit inferences about regional responses using fMRI with the temporal resolution provided by electrophysiology.     

Transmission of donor illness by stem cell transplantation: should screening be different in older donors?

With increasing donor age, the potential of transmitting diseases from donor to recipient reaches new dimensions. Potentially transmittable diseases from donors include infections, congenital disorders, and acquired illnesses like autoimmune diseases or malignancies of hematological or nonhematological origin. While established nonmalignant or malignant diseases might be easy to discover, early-stage hematological diseases like CML, light-chain multiple myelomas, aleukemic leukemias, occult myelodysplastic syndromes and other malignant and nonmalignant diseases might not be detectable by routine screening but only by invasive, new and/or expensive diagnostic tests. In the following article, we propose recommendations for donor work-up, taking into consideration the age of the donors. In contrast to blood transfusions, stem cells from donors with abnormal findings might still be acceptable for HCT, when no other options are available and life expectancy is limited. This issue is discussed in detail in relation to the available donor and stem cell source. Finally, the recommendations presented here aim at harmonized worldwide work-up for donors to insure high standard quality.     

Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines: An update comprising flunitrazolam,norflurazepam, and 4'‐chlorodiazepam (Ro5–4864)

The number of newly appearing benzodiazepine derivatives on the new psychoactive substances (NPS) drug market has increased over the last couple of years totaling 23 ‘designer benzodiazepines’ monitored at the end of 2017 by the European Monitoring Centre for Drugs and Drug Addiction. In the present study, three benzodiazepines [flunitrazolam, norflurazepam, and 4′‐chlorodiazepam (Ro5–4864)] offered as ‘research chemicals' on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by gas chromatography?mass spectrometry (GC–MS), liquid chromatography?tandem mass spectrometry (LC MS/MS), liquid chromatography?quadrupole time of flight?mass spectrometry (LC?QTOF?MS) analysis and nuclear magnetic resonance (NMR) spectroscopy. The metabolic steps of flunitrazolam were monohydroxylation, dihydroxylation, and reduction of the nitro function. The detected in vitro phase I metabolites of norflurazepam were hydroxynorflurazepam and dihydroxynorflurazepam. 4’‐Chlorodiazepam biotransformation consisted of N‐dealkylation and hydroxylation. It has to be noted that 4′‐chlorodiazepam and its metabolites show almost identical LC–MS/MS fragmentation patterns to diclazepam and its metabolites (delorazepam, lormetazepam, and lorazepam), making a sufficient chromatographic separation inevitable. Sale of norflurazepam, the metabolite of the prescribed benzodiazepines flurazepam and fludiazepam, presents the risk of incorrect interpretation of analytical findings.     

The effects of background noise on dichotic listening to consonant-vowel syllables: An fMRI study

The present fMRI study attempts to identify brain areas that may underlie the effect of different background noises on functional brain asymmetry in a dichotic listening task. Previous studies have shown that the prominent right ear advantage in dichotic listening to consonant-vowel syllables is affected by background noise. To explore the underlying neuronal processes, haemodynamic brain responses using fMRI were recorded while participants performed the dichotic listening task in two different noisy backgrounds (conversational “babble” and traffic noise). The behavioural results showed a reduction of the right ear advantage in the background noise conditions, especially in the traffic noise condition. The behavioural results are discussed in terms of alertness-attentional mechanisms. The effects of background noise on brain activation involved significant activations in a speech-processing network. Specifically the changes in activations in the peri-Sylvian region of the superior temporal gyrus and in the temporo-parietal junction part in the left hemisphere, as well as in the superior temporal gyrus/sulcus area in the right hemisphere may mirror the effects of noise on behavioural performance. The effects of noise on brain activation are discussed with regard to pre-activation mechanisms.     

5F-Cumyl-PINACA in ‘e-liquids’ for electronic cigarettes: comprehensive characterization of a new type of synthetic cannabinoid in a trendy product including investigations on the in vitro and in vivo phase I metabolism of 5F-Cumyl-PINACA and its non-fluorinated analog Cumyl-PINACA

Purpose

In recent years e-liquids used in electronic cigarettes have become an attractive alternative to smoking tobacco. A new trend is the use of e-liquids containing synthetic cannabinoids (SCs) instead of smoking cannabis or herbal mixtures laced with SCs. In the frame of a systematic monitoring of the online market of ‘legal high’ products, e-liquids from online retailers who also sell herbal blends were bought.

Methods

The products were analyzed by gas chromatography-mass spectrometry. In some of the e-liquids an unknown compound was detected which was identified as the SC 5F-Cumyl-PINACA (1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide) by nuclear magnetic resonance analysis. To investigate the phase I metabolism of this new class of compounds, 5F-Cumyl-PINACA and its non-fluorinated analog Cumyl-PINACA were incubated with pooled human liver microsomes (pHLM). Cumyl-PINACA was additionally ingested orally (0.6 mg) by a volunteer in a controlled self-experiment. To assess the relative potency of Cumyl-PINACA a set of SCs were characterized using a cAMP assay.

Results

Metabolism of 5F-Cumyl-PINACA and Cumyl-PINACA showed similarities with AM-2201 and JWH-018. The main metabolites were formed by hydroxylation at the N-pentyl side chain. The main metabolites detected in the volunteer’s urine sample were the same as in the pHLM assay. All SCs tested with the cAMP assay were full agonists at the CB₁ receptor. Cumyl-PINACA was the most potent SC among the tested compounds and showed an EC₅₀ value of 0.06 nM.

Conclusions

The increasing popularity of e-liquids particularly among young people, and the extreme potency of the added SCs, pose a serious threat to public health. To our knowledge, this is the first report describing the tentative identification of human in vivo metabolites of Cumyl-PINACA and 5F-Cumyl-PINACA.

     

Investigation of the in vitro toxicological properties of the synthetic cannabimimetic drug CP-47,497-C8

Cannabicyclohexanol (CP-47,497-C8) is a representative of a group of cannabimimetic cyclohexylphenols which is added to herbal mixtures as a cannabis substitute since 2008. Although in the beginning CP-47,497-C8 was the main ingredient of “Spice” and similar products, it was partly replaced by aminoalkylindole-type cannabinoid receptor agonists like JWH-018, JWH-073 or JWH-250, but never completely disappeared from the market. Since information on its toxicological properties is scarce, we investigated the effects of the drug in human derived cell lines. The cytotoxic effects were studied in a panel of assays (SRB, XTT, LDHe and NR tests) in a buccal derived (TR146) and a liver derived (HepG2) cell line. The strongest effects were seen in the two former assays at levels ≥ 7.5 μM indicating that the compound interferes with protein synthesis and causes membrane damage. In additional comet assays, DNA damage was detected at levels ≥ 10 μM. Experiments with lesion specific enzymes showed that these effects are not due to oxidative damage of DNA bases. The negative findings obtained in Salmonella/microsome assays and the positive results of micronucleus tests with the cell lines indicate that the compound does not cause gene mutations but acts on the chromosomal level. In contrast to other synthetic cannabinoids, no indication for estrogenic/antiestrogenic properties was seen in a luciferase assay with bone marrow derived U2-OS cells. In conclusion, our findings show that the drug has only weak cytotoxic properties. However, the induction of chromosomal damage indicates that it may cause adverse effects in users due to its impact on the stability of the genetic material.