Comparison of Extraction Methods for the Detection of Tick-Borne Encephalitis Virus RNA in Goat Raw Milk and Cream Cheese.

Infection with the tick-borne encephalitis virus (TBEV) can cause meningitis, meningoencephalitis and myelitis in humans. TBEV is an enveloped RNA virus of the family Flaviviridae, which is mostly transmitted via tick bites. However, transmission by consumption of virus-contaminated goat raw milk and goat raw milk products has also been described. Only a few methods have been reported for the detection of TBEV in food so far. Here, we compare different virus extraction methods for goat raw milk and goat raw milk cream cheese and subsequent detection of TBEV-RNA by RT-qPCR. Langat virus (LGTV), a naturally attenuated TBEV strain, was used for artificial contamination experiments. Mengovirus and the human coronavirus 229E were compared to assess their suitability to serve as internal process controls. Out of three tested extraction protocols for raw milk, sample centrifugation followed by direct RNA extraction from the aqueous interphase yielded the best results, with a recovery rate (RR) of 31.8 ± 4.9% for LGTV and a detection limit of 6.7 × 103 LGTV genome copies/ml. Out of two methods for cream cheese, treatment of the samples with TRI Reagent® and chloroform prior to RNA extraction showed the best RR of 4.7 ± 1.6% for LGTV and a detection limit of 9.4 × 104 LGTV genome copies/g. RRs of Mengovirus and LGTV were similar for both methods; therefore, Mengovirus is suggested as internal process control virus. The developed methods may be useful for screening or surveillance studies, as well as in outbreak investigations.

Myeloid-Derived Suppressor Cells Restrain Natural Killer Cell Activity in Acute Coxsackievirus B3-Induced Myocarditis.

Murine models of coxsackievirus B3 (CVB3)-induced myocarditis well represent the different outcomes of this inflammatory heart disease. Previously, we found that CVB3-infected A.BY/SnJ mice, susceptible for severe acute and chronic myocarditis, have lower natural killer (NK) cell levels than C57BL/6 mice, with mild acute myocarditis. There is evidence that myeloid-derived suppressor cells (MDSC) may inhibit NK cells, influencing the course of myocarditis. To investigate the MDSC/NK interrelationship in acute myocarditis, we used CVB3-infected A.BY/SnJ mice. Compared to non-infected mice, we found increased cell numbers of MDSC in the spleen and heart of CVB3-infected A.BY/SnJ mice. In parallel, S100A8 and S100A9 were increased in the heart, spleen, and especially in splenic MDSC cells compared to non-infected mice. In vitro experiments provided evidence that MDSC disrupt cytotoxic NK cell function upon co-culturing with MDSC. MDSC-specific depletion by an anti-Ly6G antibody led to a significant reduction in the virus load and injury in hearts of infected animals. The decreased cardiac damage in MDSC-depleted mice was associated with fewer Mac3+ macrophages and CD3+ T lymphocytes and a reduced cardiac expression of S100A8, S100A9, IL-1ß, IL-6, and TNF-α. In conclusion, impairment of functional NK cells by MDSC promotes the development of chronic CVB3 myocarditis in A.BY/SnJ mice.

Modulation of the acute defence reaction by eplerenone prevents cardiac disease progression in viral myocarditis.

AIMS: Left ventricular (LV) dysfunction in viral myocarditis is attributed to myocardial inflammation and fibrosis, inducing acute and long-time cardiac damage. Interventions are not established. On the basis of the link between inflammation, fibrosis, aldosterone, and extracellular matrix regulation, we aimed to investigate the effect of an early intervention with the mineralocorticoid receptor antagonist (MRA) eplerenone on cardiac remodelling in a murine model of persistent coxsackievirus B3 (CVB3)-induced myocarditis. METHODS AND RESULTS: SWR/J mice were infected with 5 × 104 plaque-forming units of CVB3 (Nancy strain) and daily treated either with eplerenone (200 mg/kg body weight) or with placebo starting from Day 1. At Day 8 or 28 post infection, mice were haemodynamically characterized and subsequently sacrificed for immunohistological and molecular biology analyses. Eplerenone did not influence CVB3 load. Already at Day 8, 1.8-fold (P < 0.05), 1.4-fold (P < 0.05), 3.2-fold (P < 0.01), and 2.1-fold (P < 0.001) reduction in LV intercellular adhesion molecule 1 expression, presence of monocytes/macrophages, oxidative stress, and apoptosis, respectively, was observed in eplerenone-treated vs. untreated CVB3-infected mice. In vitro, eplerenone led to 1.4-fold (P < 0.01) and 1.2-fold (P < 0.01) less CVB3-induced cardiomyocyte oxidative stress and apoptosis. Furthermore, collagen production was 1.1-fold (P < 0.05) decreased in cardiac fibroblasts cultured with medium of eplerenone-treated vs. untreated CVB3-infected HL-1 cardiomyocytes. These ameliorations were in vivo translated into prevention of cardiac fibrosis, as shown by 1.4-fold (P < 0.01) and 2.1-fold (P < 0.001) lower collagen content in the LV of eplerenone-treated vs. untreated CVB3-infected mice at Days 8 and 28, respectively. This resulted in an early and long-lasting improvement of LV dimension and function, as indicated by reduced LV end-systolic volume and end-diastolic volume, and an increase in LV contractility (dP/dtmax ) and LV relaxation (dP/dtmin ), respectively (P < 0.05). CONCLUSIONS: Early intervention with the MRA eplerenone modulates the acute host and defence reaction and prevents cardiac disease progression in experimental CVB3-induced myocarditis without aggravation of viral load. The findings advocate for an initiation of therapy of viral myocarditis as early as possible, even before the onset of inflammation-induced myocardial dysfunction. This may also have implications for coronavirus disease-19 therapy.

Serum alarmin S100A8/S100A9 levels and its potential role as biomarker in myocarditis.

AIMS: The alarmin S100A8/S100A9 (S100A8/A9) is released by activated monocytes/macrophages and neutrophils in the setting lymphocytic myocarditis (MC). We recently demonstrated its therapeutic potential in experimental acute MC. Now, we investigated the diagnostic relevance of S100A8/A9 serum levels in patients with suspected acute and chronic MC and in patients with heart failure without cardiac inflammation. METHODS AND RESULTS: Serum S100A8/A9 levels were analysed in patients with a recent onset of MC [≤ 30 days, n = 32; ejection fraction (EF): 45.4 ± 12.9%], dilated cardiomyopathy patients with inflammation (n = 112; EF: 29.0 ± 11.4%), or without inflammation (n = 58; EF: 26.6 ± 9.3%), and controls (n = 25; EF: 68.5 ± 4.6%), by using specific ELISAs. Blood samples were collected at Time Point 1 (T1), where also endomyocardial biopsies (EMBs) were withdrawn. Patients with a recent onset of MC showed a 4.6-fold increase in serum S100A8/A9 levels vs. controls (MC: 1948 ± 1670 ng/mL vs. controls: 426 ± 307 ng/mL; P < 0.0001). Serum S100A8/A9 correlated with the disease activity, represented by EMB-derived counts of inflammatory cells (CD3: r = 0.486, P = 0.0047, lymphocyte function-associated antigen-1: r = 0.558, P = 0.0009, macrophage-1 antigen: r = 0.434, P = 0.013), the EMB mRNA levels of S100A8, S100A9 (r = 0.541, P = 0.002), and left ventricular ejection fraction (LVEF: r = 0.498, P = 0.0043). EMB immunofluorescence co-stainings display macrophages/monocytes and neutrophils as the main source of S100A8 and S100A9 in recent onset MC. The diagnostic value of serum alarmin levels (cut-off 583 ng/mL) was characterized by a specificity of 92%, a sensitivity of 90.6%, positive predictive value of 93.5%, negative predictive value of 88.5%, and an accuracy of 0.949 (95% confidence interval [0.89-1]). In a subgroup of MC patients, S100A8/A9 serum levels and EMBs at T1 (n = 12) and a follow-up visit (T2, n = 12, mean follow-up 8.5 months) were available. A fall of serum S100A8/A9 (T1: 2208 ± 1843 ng/mL vs. T2: 888.8 ± 513.7 ng/mL; P = 0.00052) was associated with a reduced cardiac inflammation (CD3 T1: 70.02 ± 107.4 cells per square millimetre vs. T2: 59.18 ± 182.5 cells per square millimetre; P = 0.0342, lymphocyte function-associated antigen-1 T1: 133.5 ± 187.1 cells per square millimetre vs. T2: 74.12 ± 190.5 cells per square millimetre; P = 0.0186, and macrophage-1 antigen T1: 132.6 ± 129.5 cells per square millimetre vs. T2: 54.41 ± 65.16 cells per square millimetre; P = 0.0015). Serum S100A8/A9 levels were only slightly increased in patients within the chronic phase of MC and in heart failure patients without inflammation vs. controls. CONCLUSIONS: Serum S100A8/A9 might serve as an additional tool in the diagnostic workup of suspected acute MC patients.

Immunomodulation by adoptive regulatory T-cell transfer improves Coxsackievirus B3-induced myocarditis.

Regulatory T (Treg) cells offer new therapeutic options for controlling undesired systemic and local immune responses. The aim of the current study was to determine the impact of therapeutic Treg administration on systemic and cardiac inflammation and remodeling in coxsackievirus B3 (CVB3) -induced myocarditis. Therefore, syngeneic Treg cells were applied intravenously in CVB3-infected mice 3 d after infection. Compared with CVB3 + PBS mice, CVB3 + Treg mice exhibited lower left ventricular (LV) chemokine expression, accompanied by reduced cardiac presence of proinflammatory Ly6ChighCCR2highCx3Cr1low monocytes and higher retention of proinflammatory Ly6CmidCCR2highCx3Cr1low monocytes in the spleen. In addition, splenic myelopoiesis was reduced in CVB3 + Treg compared with CVB3 + PBS mice. Coculture of Treg cells with splenocytes isolated from mice 3 d post-CVB3 infection further demonstrated the ability of Treg cells to modulate monocyte differentiation in favor of the anti-inflammatory Ly6ClowCCR2lowCx3Cr1high subset. Treg-mediated immunomodulation was paralleled by lower collagen 1 protein expression and decreased levels of soluble and insoluble collagen in LV of CVB3 + Treg compared with CVB3 + PBS mice. In agreement with these findings, LV systolic and diastolic function was improved in CVB3 + Treg mice compared with CVB3 + PBS mice. In summary, adoptive Treg transfer in the inflammatory phase of viral-induced myocarditis protects the heart against inflammatory damage and fibrosis via modulation of monocyte subsets.-Pappritz, K., Savvatis, K., Miteva, K., Kerim, B., Dong, F., Fechner, H., Müller, I., Brandt, C., Lopez, B., González, A., Ravassa, S., Klingel, K., Diez, J., Reinke, P., Volk, H.-D., Van Linthout, S., Tschöpe, C. Immunomodulation by adoptive regulatory T-cell transfer improves Coxsackievirus B3-induced myocarditis.

Mesenchymal stromal cells inhibit NLRP3 inflammasome activation in a model of Coxsackievirus B3-induced inflammatory cardiomyopathy.

Inflammation in myocarditis induces cardiac injury and triggers disease progression to heart failure. NLRP3 inflammasome activation is a newly identified amplifying step in the pathogenesis of myocarditis. We previously have demonstrated that mesenchymal stromal cells (MSC) are cardioprotective in Coxsackievirus B3 (CVB3)-induced myocarditis. In this study, MSC markedly inhibited left ventricular (LV) NOD2, NLRP3, ASC, caspase-1, IL-1ß, and IL-18 mRNA expression in CVB3-infected mice. ASC protein expression, essential for NLRP3 inflammasome assembly, increased upon CVB3 infection and was abrogated in MSC-treated mice. Concomitantly, CVB3 infection in vitro induced NOD2 expression, NLRP3 inflammasome activation and IL-1ß secretion in HL-1 cells, which was abolished after MSC supplementation. The inhibitory effect of MSC on NLRP3 inflammasome activity in HL-1 cells was partly mediated via secretion of the anti-oxidative protein stanniocalcin-1. Furthermore, MSC application in CVB3-infected mice reduced the percentage of NOD2-, ASC-, p10- and/or IL-1ß-positive splenic macrophages, natural killer cells, and dendritic cells. The suppressive effect of MSC on inflammasome activation was associated with normalized expression of prominent regulators of myocardial contractility and fibrosis to levels comparable to control mice. In conclusion, MSC treatment in myocarditis could be a promising strategy limiting the adverse consequences of cardiac and systemic NLRP3 inflammasome activation.

Pathogenic Role of the Damage-Associated Molecular Patterns S100A8 and S100A9 in Coxsackievirus B3-Induced Myocarditis.

BACKGROUND: The alarmins S100A8 and S100A9 are damage-associated molecular patterns, which play a pivotal role in cardiovascular diseases, inflammation, and viral infections. We aimed to investigate their role in Coxsackievirus B3 (CVB3)-induced myocarditis. METHODS AND RESULTS: S100A8 and S100A9 mRNA expression was 13.0-fold (P=0.012) and 5.1-fold (P=0.038) higher in endomyocardial biopsies from patients with CVB3-positive myocarditis compared with controls, respectively. Elimination of CVB3 led to a downregulation of these alarmins. CVB3-infected mice developed an impaired left ventricular function and displayed an increased left ventricular S100A8 and S100A9 protein expression versus controls. In contrast, CVB3-infected S100A9 knockout mice, which are also a complete knockout for S100A8 on protein level, showed an improved left ventricular function, which was associated with a reduced cardiac inflammatory and oxidative response, and lower CVB3 copy number compared with wild-type CVB3 mice. Exogenous application of S100A8 to S100A9 knockout CVB3 mice induced a severe myocarditis similar to wild-type CVB3 mice. In CVB3-infected HL-1 cells, S100A8 and S100A9 enhanced oxidative stress and CVB3 copy number compared with unstimulated infected cells. In CVB3-infected RAW macrophages, both alarmins increased MIP-2 (macrophage inflammatory protein-2) chemokine expression, which was reduced in CVB3 S100A8 knockdown versus scrambled siRNA CVB3 cells. CONCLUSIONS: S100A8 and S100A9 aggravate CVB3-induced myocarditis and might serve as therapeutic targets in inflammatory cardiomyopathies.

Placenta-Derived Adherent Stromal Cells Improve Diabetes Mellitus-Associated Left Ventricular Diastolic Performance.

Left ventricular (LV) diastolic dysfunction is among others attributed to cardiomyocyte stiffness. Mesenchymal stromal cells (MSC) have cardiac-protective properties. We explored whether intravenous (i.v.) application of PLacenta-eXpanded (PLX) MSC-like cells (PLX) improves LV diastolic relaxation in streptozotocin (STZ)-induced diabetic mice and investigated underlying mechanisms. Diabetes mellitus was induced by STZ application (50 mg/kg body weight) during five subsequent days. One week after the first STZ injection, PLX or saline were i.v. applied. Two weeks later, mice were hemodynamically characterized and sacrificed. At this early stage of diabetic cardiomyopathy with low-grade inflammation and no cardiac fibrosis, PLX reduced LV vascular cell adhesion molecule-1, transforming growth factor-ß1, and interferon-γ mRNA expression, induced the percentage of circulating regulatory T cells, and decreased the splenic pro-fibrotic potential in STZ mice. STZ + PLX mice exhibited higher LV vascular endothelial growth factor mRNA expression and arteriole density versus STZ mice. In vitro, hyperglycemic PLX conditioned medium restored the hyperglycemia-impaired tube formation and adhesion capacity of human umbelical vein endothelial cells (HUVEC) via increasing nitric oxide (NO) bioavailability. PLX further induced the diabetes-downregulated activity of the NO downstream protein kinase G, as well as of protein kinase A, in STZ mice, which was associated with a raise in phosphorylation of the titin isoforms N2BA and N2B. Concomitantly, the passive force was lower in single isolated cardiomyocytes from STZ + PLX versus from STZ mice, which led to an improvement of LV diastolic relaxation. We conclude that i.v. PLX injection improves diabetes mellitus-associated diastolic performance via decreasing cardiomyocyte stiffness. Stem Cells Translational Medicine 2017;6:2135-2145.

NOD2 (Nucleotide-Binding Oligomerization Domain 2) Is a Major Pathogenic Mediator of Coxsackievirus B3-Induced Myocarditis.

BACKGROUND: The cytoplasmatic pattern recognition receptor, NOD2 (nucleotide-binding oligomerization domain 2), belongs to the innate immune system and is among others responsible for the recognition of single-stranded RNA. With Coxsackievirus B3 (CVB3) being a single-stranded RNA virus, and the recent evidence that the NOD2 target, NLRP3 (NOD-like receptor family, pyrin domain containing 3) is of importance in the pathogenesis of CVB3-induced myocarditis, we aimed to unravel the role of NOD2 in CVB3-induced myocarditis. METHODS AND RESULTS: Endomyocardial biopsy NOD2 mRNA expression was higher in CVB3-positive patients compared with patients with myocarditis but without evidence of persistent CVB3 infection. Left ventricular NOD2 mRNA expression was also induced in CVB3-induced myocarditis versus healthy control mice. NOD2 knockdown(-/-) mice were rescued from the detrimental CVB3-mediated effects as shown by a reduced cardiac inflammation (less cardiac infiltrates and suppression of proinflammatory cytokines), cardiac fibrosis, apoptosis, lower CAR (Coxsackievirus and adenovirus receptor) expression and CVB3 copy number, and an improved left ventricular function in NOD2-/- CVB3 mice compared with wild-type CVB3 mice. In agreement, NOD2-/- decreased the CVB3-induced inflammatory response, CVB3 copy number, and apoptosis in vitro. NOD2-/- was further associated with a reduction in CVB3-induced NLRP3 expression and activity as evidenced by lower ASC (apoptosis-associated speck-like protein containing a CARD) expression, caspase 1 activity, or IL-1ß (interleukin-1ß) protein expression under in vivo and in vitro CVB3 conditions. CONCLUSIONS: NOD2 is an important mediator in the viral uptake and inflammatory response during the pathogenesis of CVB3 myocarditis.

CX3CR1 knockout aggravates Coxsackievirus B3-induced myocarditis.

Studies on inflammatory disorders elucidated the pivotal role of the CX3CL1/CX3CR1 axis with respect to the pathophysiology and diseases progression. Coxsackievirus B3 (CVB3)-induced myocarditis is associated with severe cardiac inflammation, which may progress to heart failure. We therefore investigated the influence of CX3CR1 ablation in the model of acute myocarditis, which was induced by inoculation with 5x105 plaque forming units of CVB3 (Nancy strain) in either CX3CR1-/- or C57BL6/j (WT) mice. Seven days after infection, myocardial inflammation, remodeling, and titin expression and phosphorylation were examined by immunohistochemistry, real-time PCR and Pro-Q diamond stain. Cardiac function was assessed by tip catheter. Compared to WT CVB3 mice, CX3CR1-/- CVB3 mice exhibited enhanced left ventricular expression of inflammatory cytokines and chemokines, which was associated with an increase of immune cell infiltration/presence. This shift towards a pro-inflammatory immune response further resulted in increased cardiac fibrosis and cardiomyocyte apoptosis, which was reflected by an impaired cardiac function in CX3CR1-/- CVB3 compared to WT CVB3 mice. These findings demonstrate a cardioprotective role of CX3CR1 in CVB3-infected mice and indicate the relevance of the CX3CL1/CX3CR1 system in CVB3-induced myocarditis.

Human Endomyocardial Biopsy Specimen-Derived Stromal Cells Modulate Angiotensin II-Induced Cardiac Remodeling.

: Cardiac-derived adherent proliferating cells (CardAPs) are cells derived from human endomyocardial biopsy specimens; they share several properties with mesenchymal stromal cells. The aims of this study were to evaluate whether intramyocardial injection of CardAPs modulates cardiac fibrosis and hypertrophy in a mouse model of angiotensin II (Ang II)-induced systolic heart failure and to analyze underlying mechanisms. Intramyocardial application of 200,000 CardAPs improved left ventricular function. This was paralleled by a decline in left ventricular remodeling, as indicated by a reduction in cardiac fibrosis and hypertrophy. CardAPs reduced the ratio of the left ventricle to body weight and cardiac myosin expression (heavy chain), and decreased the Ang II-induced phosphorylation state of the cardiomyocyte hypertrophy mediators Akt, extracellular-signal regulated kinase (ERK) 1, and ERK2. In accordance with the antifibrotic and antihypertrophic effects of CardAPs shown in vivo, CardAP supplementation with cardiac fibroblasts decreased the Ang II-induced reactive oxygen species production, α-SMA expression, fibroblast proliferation, and collagen production. Coculture of CardAPs with HL-1 cardiomyocytes downregulated the Ang II-induced expression of myosin in HL-1. All antifibrotic and antihypertrophic features of CardAPs were mediated in a nitric oxide- and interleukin (IL)-10-dependent manner. Moreover, CardAPs induced a systemic immunomodulation, as indicated by a decrease in the activity of splenic mononuclear cells and an increase in splenic CD4CD25FoxP3, CD4-IL-10, and CD8-IL-10 T-regulatory cells in Ang II mice. Concomitantly, splenocytes from Ang II CardAPs mice induced less collagen in fibroblasts compared with splenocytes from Ang II mice. We conclude that CardAPs improve Ang II-induced cardiac remodeling involving antifibrotic and antihypertrophic effects via paracrine actions and immunomodulatory properties. SIGNIFICANCE: Despite effective pharmacological treatment with angiotensin II type I receptor antagonists or angiotensin II-converting enzyme inhibitors, morbidity and mortality associated with heart failure are still substantial, prompting the search of novel therapeutic strategies. There is accumulating evidence supporting the use of cell therapy for cardiac repair. This study demonstrates that cells derived from human endomyocardial biopsies, cardiac-derived adherent proliferating cells (CardAPs), have the potential to reduce angiotensin II-induced cardiac remodeling and improve left ventricular function in angiotensin II mice. The mechanism involves antifibrotic and antihypertrophic effects via paracrine actions and immunomodulatory properties. These findings support the potential of CardAPs for the treatment of heart failure.

Interleukin-6 receptor inhibition modulates the immune reaction and restores titin phosphorylation in experimental myocarditis.

Increased levels of interleukin-6 (IL-6) have been observed in patients with acute myocarditis and are associated with poor prognosis. This study was designed to examine whether treatment with anti-IL-6 receptor antibody improves cardiac dysfunction and left ventricular (LV) remodeling in experimental Coxsackie virus B3 (CVB3)-induced myocarditis. C57BL6/J mice were subjected to acute CVB3 infection. One day after viral infection mice were treated with a single injection of an anti-IL-6 receptor antibody (MR16-1, tocilizumab) or control IgG. Seven days after viral infection, LV function was examined by conductance catheter technique, cardiac remodeling assessed by estimation of titin phosphorylation, cardiac fibrosis, and inflammatory and antiviral response by immunohistochemistry, RT-PCR and cell culture experiments. Compared to controls, infected mice displayed an impaired systolic and diastolic LV function associated with an increase in cardiac inflammation, fibrosis and impaired titin phosphorylation. IL-6 receptor blockade led to a shift of the immune response to a Th1 direction and significant reduction of viral load. In addition, cardiac immune response, extracellular matrix regulation and titin function improved, resulting in a preserved LV function. IL-6 receptor blockade exerts cardiac beneficial effects by antiviral and immunomodulatory actions after induction of an acute murine CVB3 virus myocarditis.

Screening for illicit and medicinal drugs in whole blood using fully automated SPE and ultra-high-performance liquid chromatography with TOF-MS with data-independent acquisition.

A broad forensic screening method for 256 analytes in whole blood based on a fully automated SPE robotic extraction and ultra-high-performance liquid chromatography (UHPLC) with TOF-MS with data-independent acquisition has been developed. The limit of identification was evaluated for all 256 compounds and 95 of these compounds were validated with regard to matrix effects, extraction recovery, and process efficiency. The limit of identification ranged from 0.001 to 0.1 mg/kg, and the process efficiency exceeded 50% for 73 of the 95 analytes. As an example of application, 1335 forensic traffic cases were analyzed with the presented screening method. Of these, 992 cases (74%) were positive for one or more traffic-relevant drugs above the Danish legal limits. Commonly abused drugs such as amphetamine, cocaine, and frequent types of benzodiazepines were the major findings. Nineteen less frequently encountered drugs were detected e.g. buprenorphine, butylone, cathine, fentanyl, lysergic acid diethylamide, m-chlorophenylpiperazine, 3,4-methylenedioxypyrovalerone, mephedrone, 4-methylamphetamine, p-fluoroamphetamine, and p-methoxy-N-methylamphetamine. In conclusion, using UHPLC-TOF-MS screening with data-independent acquisition resulted in the detection of common drugs of abuse as well as new designer drugs and more rarely occurring drugs. Thus, TOF-MS screening of blood samples constitutes a practical way for screening traffic cases, with the exception of δ-9-tetrahydrocannabinol, which should be handled in a separate method.

Toxicological screening of basic drugs in whole blood using UPLC-TOF-MS.

Ultra performance liquid chromatography (UPLC) coupled with time-of-flight (TOF) mass spectrometry (MS) was established for toxicological screening of basic drugs in whole blood and tested on authentic samples. Whole blood samples (0.2 ml) were extracted using a Gilson apparatus equipped with Bond Elut Certify columns. Screening was performed for 175 compounds (psychotropic, cardiovascular, designer, and abused drugs). The drugs were separated in 15 min using a UPLC system (Waters ACQUITY BEH C18, 1.7 µm, 2.1 mm × 100 mm column) coupled to an LCT Premier XE (Waters) instrument. Data were processed by ChromaLynx XS using identification criteria of ± 0.2 min retention time, and ± 5 mDa mass tolerance. Whole blood was spiked with the 175 compounds in concentrations from 5-100 µg/kg to assess approximately the lowest concentrations that could be identified. This method was further applied to 119 samples from forensic investigations, leading to 302 hits, of which 291 (96%) were subsequently verified, in concentrations exceeding the lower limit of quantification (LLOQ), by a liquid chromatography (LC)-MS/MS confirmation method. In conclusion, this UPLC-TOF-MS method is a useful and effective screening method for basic drugs in whole blood.

Identification of ten new designer drugs by GC-MS, UPLC-QTOF-MS, and NMR as part of a police investigation of a Danish internet company.

The ability of forensic laboratories to detect and identify unknown compounds is highly important since new, non-controlled designer drugs are appearing on the market with increasing frequency. In this study, the combined use of gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC-QTOF-MS) was used for screening of new unknowns. In one large seizure from a Danish Internet company, ten different drugs were identified. Several of the compounds were seized for the first time in Denmark. The GC-MS and UPLC-QTOF-MS analyses were supplemented by nuclear magnetic resonance (NMR) spectra for the structural elucidation of p-fluoroamphetamine, mephedrone (4-methylmethcathinone), flephedrone (4-fluoromethcathinone), PPP (α-pyrrolidinopropiophenone), MDPV (3,4-methylenedioxypyrovalerone), Bk-MBDB (2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one), pFBT (3-(pfluorobenzoyl)-tropane), and JWH-073 (1-butyl-3-(1-naphthoyl)indol), whereas methylone (3,4-methylenedioxymethcathinone) and N-ethylcathinone matched electron impact-mass spectrometry (EI-MS) library spectra and therefore the screenings were considered sufficient. EI-MS spectra and the proposed main fragmentation patterns are presented as well as QTOF-MS exact masses and fragments and NMR chemical shifts. For the ß-ketophenylethylamines (mephedrone, flephedrone, PPP, MDPV, Bk-MBDB, methylone, and N-ethylcathinone) some general fragmentation patterns observed in the EI-MS and QTOF-MS spectra are further discussed and compared to other ß-ketophenylethylamines.

The use of massage therapy for reducing pain, anxiety, and depression in oncological palliative care patients: a narrative review of the literature.

A considerable number of cancer patients use complementary medicine therapies in order to alleviate different symptoms such as pain, anxiety, and depression, occurring in connection with cancer. This paper explores the question to what extent massage therapies are able to reduce the amount of pain, anxiety, and depression. For this purpose, a systematic literature analysis was carried out in the electronic databases and specialist journals. There is already evidence that massage therapies can influence the symptoms of pain, anxiety, and depression in a positive way.

Drug-related death in Denmark in 2007.

INTRODUCTION: We investigated fatal poisonings among drug addicts in 2007. The cause of death, abuse pattern and geographic differences are presented. MATERIAL AND METHODS: All drug-related deaths examined at the three forensic medicine institutes in Denmark in 2007 were evaluated. RESULTS: The number of drug-related deaths in 2007 was 226. Methadone deaths had increased since 1997 while heroin/morphine deaths decreased. In earlier studies, very few deaths from central stimulants like cocaine and amphetamines occurred (1-1.5%), but in 2007 6% of the deaths were caused by these drugs. Multiple drug use was common. Heroin/morphine, cocaine, amphetamine, cannabis, methadone, benzodiazepines and alcohol were included in the poly-drug use. CONCLUSION: This investigation shows stabilization in the number of fatal poisonings in drug addicts. Geographic differences were observed. Methadone was the most frequent cause of fatal poisoning and there was a continuous decrease in heroin/morphine deaths. Fatal deaths from cocaine and amphetamine have increased considerably. Multiple drug use was common. FUNDING: not relevant. TRIAL REGISTRATION: not relevant.

1-Alkyl-4-phenyl-6-alkoxy-1H-quinazolin-2-ones: a novel series of potent calcium-sensing receptor antagonists.

Parathyroid hormone (PTH) is an effective bone anabolic agent. However, only when administered by daily sc injections exposure of short duration is achieved, a prerequisite for an anabolic response. Instead of applying exogenous PTH, mobilization of endogenous stores of the hormone can be envisaged. The secretion of PTH stored in the parathyroid glands is mediated by a calcium sensing receptor (CaSR) a GPCR localized at the cell surface. Antagonists of CaSR (calcilytics) mimic a state of hypocalcaemia and stimulate PTH release to the bloodstream. Screening of the internal compound collection for inhibition of CaSR signaling function afforded 2a. In vitro potency could be improved >1000 fold by optimization of its chemical structure. The binding mode of our compounds was predicted based on molecular modeling and confirmed by testing with mutated receptors. While the compounds readily induced PTH release after iv application a special formulation was needed for oral activity. The required profile was achieved by using microemulsions. Excellent PK/PD correlation was found in rats and dogs. High levels of PTH were reached in plasma within minutes which reverted to baseline in about 1-2 h in both species.

Two cases of lysergamide intoxication by ingestion of seeds from Hawaiian Baby Woodrose.

We describe two cases of human consumption of seeds from Argyreia nervosa (Hawaiian Baby Woodrose), which resulted in one fatality due to falling from a building and one surviving witness. The principal psychoactive constituent of the seeds, lysergamide (LSA), was recovered from blood and urine samples by mixed-mode cation exchange solid-phase extraction and quantified by ultra performance liquid chromatography-time of flight mass spectrometry (UPLC-ToF/MS). The LSA concentrations were determined by UPLC-ToF/MS to be 4.9 microg/L in blood and 1.0mg/L in urine in the dead person and 1.8 microg/L in blood and 0.50mg/L in urine in the living person. These analytical findings were found to be in accordance with the case story, which indicated that seeds had been ingested and also noted psychological reactions, i.e. the will to jump out of the window. Other findings in the dead person were 22 microg/L THC in blood, 0.71 g/L ethanol in blood and 1.0 g/L ethanol in vitreous humor. Constituents originating from the seeds of A. nervosa, i.e. LSA, ergonovine, lysergic acid alpha-hydroxyethylamide were also identified in the biological samples. The 2-hydroxy-3-oxo metabolites of LSA and ergonovine were identified in the urine sample of the deceased.

[Massage and music therapy for relief of anxiety of cancer patients in palliative care]. / Massagen und Musiktherapie zur Reduktion der Angst von onkologischen Patienten bei der Palliativversorgung.

In palliative care between 13.9 and 25 percent of all patients suffering from cancer show signs of anxiety disorders. Up to 75 percent of these patients suffer from non-pathologic anxiety, which has a negative impact on the patients' quality of life. Therefore it is important to provide interventions that are able to reduce anxiety of cancer patients. Massage and music therapy are effective interventions for minimizing anxiety of cancer patients in palliative care. An empathic attitude of the nurse increases the effect of the mentioned interventions. While there is evidence of the interventions mentioned it is yet necessary to further explore these in additional clinical trials to consolidate the already existing results.