Metabolomic and lipidomic fingerprints in inflammatory skin diseases - Systemic illumination of atopic dermatitis, hidradenitis suppurativa and plaque psoriasis.

Auto-inflammatory skin diseases place considerable symptomatic and emotional burden on the affected and put pressure on healthcare expenditures. Although most apparent symptoms manifest on the skin, the systemic inflammation merits a deeper analysis beyond the surface. We set out to identify systemic commonalities, as well as differences in the metabolome and lipidome when comparing between diseases and healthy controls. Lipidomic and metabolomic LC-MS profiling was applied, using plasma samples collected from patients suffering from atopic dermatitis, plaque-type psoriasis or hidradenitis suppurativa or healthy controls. Plasma profiles revealed a notable shift in the non-enzymatic anti-oxidant defense in all three inflammatory disorders, placing cysteine metabolism at the center of potential dysregulation. Lipid network enrichment additionally indicated the disease-specific provision of lipid mediators associated with key roles in inflammation signaling. These findings will help to disentangle the systemic components of autoimmune dermatological diseases, paving the way to individualized therapy and improved prognosis.

Endocannabinoid analysis in GlucoEXACT plasma: Method validation and sample handling recommendations.

Endocannabinoids (ECs), such as anandamide and 2-arachidonyl glycerol (2-AG), contribute to the pathology of inflammatory, malignant, cardiovascular, metabolic and mental diseases. The reliability of quantitative analyses in biological fluids of ECs and endocannabinoid-like (EC-like) substances depends on pre-analytical conditions such as temperature and "time-to-centrifugation". Standardization of these parameters is critical for valid quantification and implementation in clinical research. In this study, we compared concentrations obtained with GlucoEXACT blood collection tubes versus K3EDTA tubes and employed the optimized procedure to assess ECs profiles in patients with inflammatory skin disease and healthy controls. A UHPLC-MS/MS method was validated for human plasma from GlucoEXACT blood collection tubes according to EMA and FDA guidelines, and pre-analytical conditions were systematically modified to assess analyte stability and optimize the procedures. The results showed significantly lower concentrations of ECs and EC-like substance concentrations with GlucoEXACT tubes compared with K3EDTA tubes, and GlucoEXACT extended the time window of stable concentrations. The strongest method-disagreement occurred for 1/2-AG suggesting that GlucoEXACT delayed ex vivo isomer rearrangement. Hence, GlucoExact tubes were superior in terms of stability and reliability. However, although absolute concentrations obtained with GlucoExact and K3EDTA differed, linear regression studies showed high agreement (except for 1/2-AG), and both methods showed similar EC profiles and similar disease-dependent pro-inflammatory patterns in dermatology patients. Hence, despite the obstacles in EC analyses, implementation of optimized pre-analytical blood collection and sample processing procedures provide reliable insight into peripheral ECs.

Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research.

The emerging disciplines of lipidomics and metabolomics show great potential for the discovery of diagnostic biomarkers, but appropriate pre-analytical sample-handling procedures are critical because several analytes are prone to ex vivo distortions during sample collection. To test how the intermediate storage temperature and storage period of plasma samples from K3EDTA whole-blood collection tubes affect analyte concentrations, we assessed samples from non-fasting healthy volunteers (n = 9) for a broad spectrum of metabolites, including lipids and lipid mediators, using a well-established LC-MS-based platform. We used a fold change-based approach as a relative measure of analyte stability to evaluate 489 analytes, employing a combination of targeted LC-MS/MS and LC-HRMS screening. The concentrations of many analytes were found to be reliable, often justifying less strict sample handling; however, certain analytes were unstable, supporting the need for meticulous processing. We make four data-driven recommendations for sample-handling protocols with varying degrees of stringency, based on the maximum number of analytes and the feasibility of routine clinical implementation. These protocols also enable the simple evaluation of biomarker candidates based on their analyte-specific vulnerability to ex vivo distortions. In summary, pre-analytical sample handling has a major effect on the suitability of certain metabolites as biomarkers, including several lipids and lipid mediators. Our sample-handling recommendations will increase the reliability and quality of samples when such metabolites are necessary for routine clinical diagnosis.

Post-mortem changes of prostanoid concentrations in tissues of mice: Impact of fast cervical dislocation and dissection delay.

Prostanoids are potent lipid mediators involved in a wide variety of physiological functions like blood pressure regulation or inflammation as well as cardiovascular and malign diseases. Elucidation of their modes of action is mainly carried out in pre-clinical animal models by quantifying prostanoids in tissues of interest. Unfortunately, prostanoids are prone to post-mortem artifact formation and de novo synthesis can already be caused by external stimuli during the euthanasia of animals like prolonged hypercapnia or ischemia. Therefore, this study investigates the suitability and impact of fast cervical dislocation for the determination of prostanoids (6-keto-PGF1α, TXB2, PGF2α, PGD2, PGE2) in seven tissues of mice (spinal cord, brain, sciatic nerve, kidney, liver, lung, and spleen) to minimize time-dependent effects and approximate physiological concentrations. Tissues were dissected in a standardized sequence directly or after 10 min to investigate the influence of dissection delays. The enzyme inhibitor indomethacin (10 µM) in combination with low processing temperatures was employed to preserve prostanoid concentrations during sample preparation. Quantification of prostanoids was performed via LC-MS/MS. This study shows, that prostanoids are differentially susceptible to post-mortem artifact formation which is closely connected to their physiological function and metabolic stability in the respective tissues. Prostanoids in the brain, spinal cord, and kidney that are not involved in the regulatory response post-mortem, i.e. blood flow regulation (6-keto-PGF1α, PGE2, PGF2α) showed high reproducibility even after dissection delay and could be assessed after fast cervical dislocation if prerequisites like standardized pre-analytical workflows with immediate dissection and inhibition of residual enzymatic activity are in place. However, in tissues with high metabolic activity (liver, lung) more stable prostanoid metabolites should be used. Moreover, prostanoids in the spleen were strongly affected by dissection delays and presumably the method of euthanasia itself.

Pre-analytical challenges for the quantification of endocannabinoids in human serum.

Endocannabinoids (ECs) are potent lipid mediators with high physiological relevance. They are involved in a wide variety of diseases like depression or multiple sclerosis and are closely connected to metabolic parameters in humans. Therefore, their suitability as a biomarker in different (patho-)physiological conditions is discussed intensively and predominantly investigated by analyzing systemic concentrations in easily accessible matrices like blood. Carefully designed pre-analytical sample handling is of major importance for high-quality data, but harmonization is not achieved yet. Whole blood is either processed to serum or plasma before the onset of analytical workflows and while knowledge about pre-analytical challenges in plasma handling is thorough they were not systematically investigated for serum. Therefore, the ECs AEA and 2-AG, and closely related EC-like substances 1-AG, DHEA, and PEA were examined by LC-MS/MS in serum samples of nine healthy volunteers employing different pre-analytical sample handling protocols, including prolonged coagulation, and storage after centrifugation at room temperature (RT) or on ice. Furthermore, all analytes were also assessed in plasma samples obtained from the same individuals at the same time points to investigate the comparability between those two blood-based matrices regarding obtained concentrations and their 2-AG/1-AG ratio. This study shows that ECs and EC-like substances in serum samples were significantly higher than in plasma and are especially prone to ex vivo changes during initial and prolonged storage for coagulation at RT. Storage on ice after centrifugation is less critical. However, storage at RT further increases 1-AG and 2-AG concentrations, while also lowering the already reduced 2-AG/1-AG ratio due to isomerization. Thus, avoidance of prolonged processing at RT can increase data quality if serum as the matrix of choice is unavoidable. However, serum preparation in itself is expected to initiate changes of physiological concentrations as standard precautionary measures like fast and cooled processing can only be utilized by using plasma, which should be the preferred matrix for analyses of ECs and EC-like substances.

Determination of endocannabinoids and endocannabinoid-like substances in human K3EDTA plasma - LC-MS/MS method validation and pre-analytical characteristics.

The determination of endocannabinoids and endocannabinoid-like substances in biological human samples is a vibrant field of research with great significance due to postulated relevance of these substances in diseases such as Alzheimer's disease, multiple sclerosis, cancer and cardiovascular diseases. For a possible use as biomarker in early prediction or diagnosis of a disease as well as examination of a successful treatment, the valid determination of the analytes in common accessible human samples, such as plasma or serum, is of great importance. A method for the determination of arachidonoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, 1-arachidonoyl glycerol and 2-arachidonoyl glycerol in human K3EDTA plasma using liquid-liquid-extraction in combination with liquid chromatography-tandem-mass spectrometry has been developed and validated for the quantification of the aforementioned analytes. Particular emphasis was placed on the chromatographic separation of the isomers 1-arachidonoyl glycerol and 2-arachidonoyl glycerol, arachidonoyl ethanolamide and O-arachidonoyl ethanolamine (virodhamine) as well as oleoyl ethanolamide and vaccenic acid ethanolamide. During the validation process, increasing concentrations of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol while storing plasma samples were observed. In-depth investigation of pre-analytical sample handling revealed rising concentrations for both analytes in plasma and for arachidonoyl ethanolamide, oleoyl ethanolamide and palmitoyl ethanolamide in whole blood, dependent on the period and temperature of storage. Prevention of the increase in concentration was not possible, raising the question whether human K3EDTA plasma is suitable for the determination of endocannabinoids and endocannabinoid-like substances. Especially the common practice to calculate the concentration of 2-arachidonoyl glycerol as sum of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol is highly questionable because the concentrations of both analytes increase unequally while storing the plasma samples in the fridge.

A validated LC-MS/MS method for the determination of homocysteic acid in biological samples.

The role of homocysteic acid (HCA) in severe diseases like Alzheimer's disease is under discussion and some recent studies correlate elevated HCA concentrations with the diagnosis of Alzheimer's. However, non-selective and insufficiently sensitive methods have been used to quantitate HCA and results of different studies show large differences in the determined HCA concentration in samples from patients and controls, and therefore non-comparable results. An accurate and precise quantitation method for the determination of HCA in human serum, urine and CSF has been developed by using a combination of protein precipitation and solid phase extraction for sample preparation followed by an LC-MS/MS analysis using a combination of a HILIC separation and tandem mass spectrometry. The developed method has been fully validated in accordance with the guidelines provided by the US Food and Drug administration FDA and the European Medicines Agency EMA. Furthermore, the method has demonstrated its ability to determine the endogenous HCA concentration in serum and urine samples from healthy volunteers.

Immunogenicity assay development and validation for biological therapy as exemplified by ustekinumab.

Introduction of biotherapeutics has been a major milestone in the treatment of different chronic diseases. Nevertheless, the immune system can recognize the administered biological as non-self and respond with generation of anti-drug antibodies (ADA), including neutralizing ADA (nADA). Immunogenic responses may result in altered drug dynamics and kinetics leading to changes in safety and efficacy. However, there are several challenges with standard techniques for immunogenicity testing. Ustekinumab (UST), used in different inflammatory diseases, is a therapeutic antibody directed against the shared p40 subunit of interleukin (IL)-12 and IL-23, interfering in the pathogenically crucial T helper type 1 (Th1)/Th17 pathway. We established and validated different approaches for detection and quantitation of UST, UST-specific ADA and nADA. Addressing the obstacle of complex formation of UST with nADA, we developed an acidification assay to approach the total amount of nADA. Validated methods were based on surface plasmon resonance spectroscopy (SPR), enzyme-linked immunosorbent assay (ELISA) and a cell-based approach to characterize neutralizing capacity of nADA. Parameters assessed were determination and quantitation limits, linearity, range, precision, accuracy and selectivity. Quantitation of ADA and UST was feasible at lower concentrations using ELISA, whereas SPR showed a wider linear range for determination of ADA and UST. Accuracy, precision and linearity for quantitation were comparable using ELISA, SPR and the cell-based approach. All validated parameters fulfill the requirements of regulatory agencies. A combination of the testing approaches could address the increasing demand of precision medicine as it can be suitable for capturing the whole spectrum of immunogenicity and is transferable to other biologicals.

Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis.

Bioactive lipids contribute to the pathophysiology of multiple sclerosis. Here, we show that lysophosphatidic acids (LPAs) are dysregulated in multiple sclerosis (MS) and are functionally relevant in this disease. LPAs and autotaxin, the major enzyme producing extracellular LPAs, were analyzed in serum and cerebrospinal fluid in a cross-sectional population of MS patients and were compared with respective data from mice in the experimental autoimmune encephalomyelitis (EAE) model, spontaneous EAE in TCR1640 mice, and EAE in Lpar2 -/- mice. Serum LPAs were reduced in MS and EAE whereas spinal cord LPAs in TCR1640 mice increased during the 'symptom-free' intervals, i.e. on resolution of inflammation during recovery hence possibly pointing to positive effects of brain LPAs during remyelination as suggested in previous studies. Peripheral LPAs mildly re-raised during relapses but further dropped in refractory relapses. The peripheral loss led to a redistribution of immune cells from the spleen to the spinal cord, suggesting defects of lymphocyte homing. In support, LPAR2 positive T-cells were reduced in EAE and the disease was intensified in Lpar2 deficient mice. Further, treatment with an LPAR2 agonist reduced clinical signs of relapsing-remitting EAE suggesting that the LPAR2 agonist partially compensated the endogenous loss of LPAs and implicating LPA signaling as a novel treatment approach. Graphical summary of lysophosphatidic signaling in multiple sclerosis.

Cleavage of SNAP-25 ameliorates cancer pain in a mouse model of melanoma.

BACKGROUND: Cancer pain is associated with increased pain sensitivity to noxious (hyperalgesia) and normally innocuous (allodynia) stimuli due to activation of nociceptors by tumour-derived mediators or tumour infiltration of nerves. The pain sensitization is accompanied by modifications in gene expression, but specifically regulated genes are largely unknown. The 25 kDa synaptosomal-associated protein (SNAP-25) is involved in chemical neurotransmission at the synaptic cleft. Its inhibition by Botulinum neurotoxin A (BoNT/A) has been associated with antinociceptive effects in migraine, inflammatory and neuropathic pain. However, its potential to reduce tumour-associated pain remains to be clarified. METHODS: We applied a melanoma model of tumour pain in C57BL/6 mice and investigated SNAP-25 expression and regulation by qRT-PCR, Western Blot and immunofluorescence as well as tumour-associated mechanical allodynia with and without BoNT/A treatment. RESULTS: We found increased SNAP-25 expression in the dorsal root ganglia and the sciatic nerve. Intraplantar injection of BoNT/A induced the cleavage of SNAP-25 in these tissues and was associated with decreased mechanical allodynia after therapeutic treatment at early and late stages of tumour pain while the tumour size was not affected. CONCLUSIONS: Our data indicate that SNAP-25 plays a role in tumour pain but has no influence on the initiation and progression of skin cancer. Its cleavage inhibits the development of allodynia in the mouse melanoma model and might be useful as new therapeutic approach for the treatment of cancer pain. WHAT DOES THIS STUDY ADD?: SNAP-25 is differentially regulated during melanoma-induced tumour pain. Its cleavage by BoNT/A might be a suitable therapeutic option for tumour pain patients since tumour-associated pain can be strongly and significantly reduced after preventive and therapeutic BoNT/A treatment, respectively.

PAFAH1B1 and the lncRNA NONHSAT073641 maintain an angiogenic phenotype in human endothelial cells.

AIM: Platelet-activating factor acetyl hydrolase 1B1 (PAFAH1B1, also known as Lis1) is a protein essentially involved in neurogenesis and mostly studied in the nervous system. As we observed a significant expression of PAFAH1B1 in the vascular system, we hypothesized that PAFAH1B1 is important during angiogenesis of endothelial cells as well as in human vascular diseases. METHOD: The functional relevance of the protein in endothelial cell angiogenic function, its downstream targets and the influence of NONHSAT073641, a long non-coding RNA (lncRNA) with 92% similarity to PAFAH1B1, were studied by knockdown and overexpression in human umbilical vein endothelial cells (HUVEC). RESULTS: Knockdown of PAFAH1B1 led to impaired tube formation of HUVEC and decreased sprouting in the spheroid assay. Accordingly, the overexpression of PAFAH1B1 increased tube number, sprout length and sprout number. LncRNA NONHSAT073641 behaved similarly. Microarray analysis after PAFAH1B1 knockdown and its overexpression indicated that the protein maintains Matrix Gla Protein (MGP) expression. Chromatin immunoprecipitation experiments revealed that PAFAH1B1 is required for active histone marks and proper binding of RNA Polymerase II to the transcriptional start site of MGP. MGP itself was required for endothelial angiogenic capacity and knockdown of both, PAFAH1B1 and MGP, reduced migration. In vascular samples of patients with chronic thromboembolic pulmonary hypertension (CTEPH), PAFAH1B1 and MGP were upregulated. The function of PAFAH1B1 required the presence of the intact protein as overexpression of NONHSAT073641, which was highly upregulated during CTEPH, did not affect PAFAH1B1 target genes. CONCLUSION: PAFAH1B1 and NONHSAT073641 are important for endothelial angiogenic function.

A small yet comprehensive subset of human experimental pain models emerging from correlation analysis with a clinical quantitative sensory testing protocol in healthy subjects.

BACKGROUND: Picturing the complexity of pain in human experimental settings has increased the predictivity for clinical pain but requires increasingly complex test batteries. This raises problems in studies in which time is objectively limited, for example by the course of action of an analgesic drug. We addressed the selection of a small yet comprehensive set of pain tests for the use in such a situation. METHOD: Nineteen different pain measures from 'classical' pain models (n = 9) and a clinically established QST-pain test battery (n = 10), were obtained from 72 healthy volunteers (34 men). The nonparametric correlation structure among the various pain measures was analysed using Ward clustering. RESULTS: Four clusters emerged, each consisting of highly correlated pain measures. The pain model groups emerged comprised (I) pain thresholds and tolerances to blunt pressure or electrical pain; (II) pain thresholds to thermal stimuli; (III) pain measures obtained following application of punctate mechanical, intranasal CO2 chemical or cutaneous laser heat stimuli; and (IV) detection thresholds to thermal stimuli. The first three clusters agreed with an immediate mechanistic interpretation as reflecting C-fibre mediated pain, thermal pain and Aδ-fibre mediated pain, respectively, whereas the last cluster contained non-painful measures and was disregarded. CONCLUSIONS: When basing a selection of a small comprehensive set of pain models on the assumption that highly correlated pain measures account for redundant results and therefore, one member of each group suffices an economic yet comprehensive pain study, results suggest inclusion of established C-fibre, Aδ-fibre mediated and thermal pain measures.

Multiple rodent models and behavioral measures reveal unexpected responses to FTY720 and DMF in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a widely-used rodent model for multiple sclerosis (MS), but a single model can hardly capture all features of MS. We investigated whether behavioral parameters in addition to clinical motor function scores could be used to assess treatment efficacy during score-free intervals in the relapsing-remitting EAE model in SJL/J mice. We studied the effects of the clinical reference compounds FTY720 (fingolimod, 0.5mg/kg/day) and dimethyl fumarate (DMF, 20-30 mg/kg/day) on clinical scores in several rodent EAE models in order to generate efficacy profiles. SJL/J mice with relapsing-remitting EAE were studied using behavioral tests, including rotarod, gait analysis, locomotor activity and grip strength. SJL/J mice were also examined according to Crawley's sociability and preference for social novelty test. Prophylactic treatment with FTY720 prevented clinical scores in three of the four EAE rodent models: Dark Agouti (DA) and Lewis rats and C57BL/6J mice. Neither prophylactic nor late-therapeutic treatment with FTY720 reduced clinical scores or reversed deficits in the rotarod test in SJL/J mice, but we observed effects on motor functions and sociability in the absence of clinical scores. Prophylactic treatment with FTY720 improved the gait of SJL/J mice whereas late-therapeutic treatment improved manifestations of reduced social (re)cognition or preference for social novelty. DMF was tested in three EAE models and did not improve clinical scores at the dose used. These data indicate that improvements in behavioral deficits can occur in absence of clinical scores, which indicate subtle drug effects and may have translational value for human MS.

[Pharmacological aspects of pain research in Germany]. / Pharmakologische Aspekte der Schmerzforschung in Deutschland.

In spite of several approved analgesics, the therapy of pain still constitutes a challenge due to the fact that the drugs do not exert sufficient efficacy or are associated with severe side effects. Therefore, the development of new and improved painkillers is still of great importance. A number of highly qualified scientists in Germany are investigating signal transduction pathways in pain, effectivity of new drugs and the so far incompletely investigated mechanisms of well-known analgesics in preclinical and clinical studies. The highlights of pharmacological pain research in Germany are summarized in this article.

[Drug interactions in pain therapy]. / Arzneimittelinteraktionen in der Schmerztherapie.

BACKGROUND: Pain is one of the most common reasons for consulting a physician. Chronic pain patients often suffer from a variety of comorbidities, such as depression and anxiety and they are therefore often simultaneously treated with more than one drug. The probability of drug interactions increases with every additional drug. MATERIAL AND METHODS: A systematic internet and literature search up to February 2015 was carried out. Systematic lists were included. In addition, the drug prescription information sheets were used and an internet search via Pubmed and google.com was carried out for drugs alone and in combination in order to find substance-specific interactions. RESULTS: A differentiation is made between pharmaceutical, pharmacodynamic and pharmacokinetic drug interactions. Pharmaceutical interactions are caused by chemical, physical or physicochemical incompatibility of drugs or adjuvants used. These can even occur outside the body and during concomitant administration via the same route. A pharmacodynamic interaction in pain management is for example the additive sedative effect of opioids and benzodiazepines when taken together. Pharmacokinetic interactions occur during the absorption, distribution, metabolism and in the elimination phases. CONCLUSION: Many drug interactions can be avoided by careful and continuous evaluation of pharmacotherapy and if necessary its adaptation; however, a sound knowledge of the underlying pharmacological mechanisms and the properties of currently used analgesics is necessary.

Nano-LC-MS/MS for the quantitation of prostanoids in immune cells.

Prostanoids, derivatives of arachidonic acid, are involved in inflammation and immune reactions. To understand the role of prostanoids produced by diverse immune cells, a highly sensitive quantitation method for prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), 6-keto prostaglandin F1α (6-keto PGF1α), prostaglandin F2α (PGF2α), and thromboxane B2 (TXB2) by means of nano-liquid chromatography-tandem mass spectrometry has been developed. It was validated according to the guidelines of the Food and Drug Administration (FDA) in terms of linearity, precision, accuracy, recovery, stability, and lower limit of quantitation (LLOQ). The LLOQ were 25 pg/mL in the injected solution (75 fg on column (o.c.)) for PGE2 and PGD2 and 37.5 pg/mL (112.5 fg on column) for 6-keto PGF1α, PGF2α, and TXB2, respectively. It was successfully applied to murine mast cells isolated from paws after zymosan injection and to CD4(+) and CD8(+) T lymphocytes from blood of sensitized versus non-sensitized mice in context of a delayed type hypersensitivity model. About 5,000 (T cells) to 40,000 (mast cells) cells were sufficient for quantitation. In the mast cells, the production of PGE2 increased at a significantly higher extent than the synthesis of the other prostanoids. The T lymphocytes did not show any difference in prostanoid production, no matter whether they were obtained from sensitized mice or non-sensitized mice.

Cytochrome P450 epoxygenase dependence of opioid analgesia: fluconazole does not interfere with remifentanil-mediated analgesia in human subjects.

Cytochrome P450 (CYP) inhibitors may reduce opioid analgesia by inhibiting CYP activity-dependent post-opioid receptor signaling pathways in the brain. This suggestion was predicated on observations of highly attenuated morphine antinociception in rodents after intracerebroventricular injection of fluconazole or carrying a neuron-specific deletion of the cytochrome P450 reductase. However, based on assessments of thermal and electrical pain tolerance, respiratory function, and side effects in 21 healthy volunteers, before and during steady-state concentrations of 1.5 and 3.0 ng/ml of remifentanil at the effect site (viz., the central nervous system), administration of 400 mg/day fluconazole for 8 days in a double-blind, placebo-controlled manner failed to attenuate opioid effects. Although CYP inhibitors such as fluconazole are unlikely to attenuate remifentanil analgesia in humans, extrapolation of the findings to other opioids is premature because differences among opioid effects, such as ligand-selective biased signaling at opioid receptors, leave the possibility that CYP-dependent opioid signaling in the brain might be limited to morphine and may not extend to remifentanil.

Biotechnological approach towards a highly efficient production of natural prostaglandins.

Prostaglandins (PGs) act as potent local hormones in nearly all tissues of the human body and are used for various medical applications. Heterologous expression of PG endoperoxide H-synthase from the alga, Gracilaria vermiculophylla, into E. coli and the application of this strain in biotransformation experiments resulted in a highly efficient conversion of arachidonic acid (ARA) yielding up to 130 mg natural PGs l(-1) in a laboratory scale approach. Detailed analyses of the products and production kinetics were performed, confirming a rapid conversion of ARA to PGs.

Bupivacaine-induced cellular entry of QX-314 and its contribution to differential nerve block.

BACKGROUND AND PURPOSE: Selective nociceptor fibre block is achieved by introducing the cell membrane impermeant sodium channel blocker lidocaine N-ethyl bromide (QX-314) through transient receptor potential V1 (TRPV1) channels into nociceptors. We screened local anaesthetics for their capacity to activate TRP channels, and characterized the nerve block obtained by combination with QX-314. EXPERIMENTAL APPROACH: We investigated TRP channel activation in dorsal root ganglion (DRG) neurons by calcium imaging and patch-clamp recordings, and cellular QX-314 uptake by MS. To characterize nerve block, compound action potential (CAP) recordings from isolated nerves and behavioural responses were analysed. KEY RESULTS: Of the 12 compounds tested, bupivacaine was the most potent activator of ruthenium red-sensitive calcium entry in DRG neurons and activated heterologously expressed TRPA1 channels. QX-314 permeated through TRPA1 channels and accumulated intracellularly after activation of these channels. Upon sciatic injections, QX-314 markedly prolonged bupivacaine's nociceptive block and also extended (to a lesser degree) its motor block. Bupivacaine's blockade of C-, but not A-fibre, CAPs in sciatic nerves was extended by co-application of QX-314. Surprisingly, however, this action was the same in wild-type, TRPA1-knockout and TRPV1/TRPA1-double knockout mice, suggesting a TRP-channel independent entry pathway. Consistent with this, high doses of bupivacaine promoted a non-selective, cellular uptake of QX-314. CONCLUSIONS AND IMPLICATIONS: Bupivacaine, combined with QX-314, produced a long-lasting sensory nerve block. This did not require QX-314 permeation through TRPA1, although bupivacaine activated these channels. Regardless of entry pathway, the greatly extended duration of block produced by QX-314 and bupivacaine may be clinically useful.

Congenital taurine deficiency in mice is associated with reduced sensitivity to nociceptive chemical stimulation.

The amino acid taurine is required for development and functioning of the central and peripheral nervous system where it exerts osmoregulatory, neuromodulatory and anti-apoptotic actions. It is subject to cellular import by the taurine transporter slc6a6. Absence of the transporter and consequently, absence of taurine leads to several neurologic deficits and sensory losses. In a slc6a6 knock-out mouse model, consequences of congenital taurine deficiency were assessed in nociceptive sensory processes. The formalin assay, hot plate assay, and summated generator potentials in response to local nociceptive stimulation with gaseous CO2 were applied. Reduced responsiveness of slc6a6(-/-) mice to nociceptive stimulation was observed in particular to chemical nociceptive stimuli. Scl6a6 knock-out mice spent significantly less time licking the formalin injected paw and displayed smaller amplitudes of the nociceptive nasal mucosa potentials than wild-type mice (p=0.002 and 0.01 respectively). In contrast, withdrawal latencies on a hot plate did not significantly differ, suggesting that intracellular taurine deficits lead in particular to a hyposensitivity of nociceptive sensory neurons sensitive to noxious chemical stimulation. As hereditary absence of taurine affects biological processes of anatomical structure development, the altered nociceptive responses likely reflect consequences of compromised peripheral nervous system development.