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Background The role of inflammatory processes in the etiology of schizophrenia is increasingly being investigated. A link between psychosis and inflammation measured with different biomarkers has been reported in the literature and needs to be further explored. To investigate the presence of inflammatory biomarkers in first-episode psychosis (FEP) we analyzed the largest available FEP cohort to date regarding routine CSF and blood diagnostics. Methods We report a retrospective analysis of clinical data from all inpatients that were admitted to our tertiary care hospital with a ICD-10 diagnosis of F2x (schizophrenia-spectrum) between January 1, 2008 and August 1, 2018 and underwent a lumbar puncture. Results A total of n = 314 FEP patients were included in our sample. 42.7% patients (134/314) showed cerebrospinal fluid (CSF) alterations. Oligoclonal bands in the CSF were present in 21.8% of patients (67/307) with 12.4% (27/217) of patients presenting OCBs type 2 or 3. 15.8% (49/310) of our cohort revealed signs of blood-brain-barrier (BBB) dysfunction with increased albumin ratios. Mean serum CRP levels were 2.4 mg/l (SD = 9.5). CRP elevation was present in 116/280 cases (41.4%). Conclusions This large retrospective analysis on FEP cohort greatly enriches the clinical data available on this population and contributes to the discussion around inflammation in psychosis. Of note, even though several inflammatory alterations were found both in CSF and in blood tests, we found no evidence for a significant relationship between peripheral inflammation and inflammatory CSF. Furthermore, no significant relationship between CSF alterations and peripheral inflammation measured with CRP could be established.

ObjectivesTo evaluate the long-term effects of natalizumab (NTZ) on different features of intrathecal immunoglobulin (Ig) synthesis in patients with multiple sclerosis (MS) and to quantify the expression of α4-integrin in stages of B-cell maturation.MethodsWe combined a cross-sectional (49 NTZ-treated MS patients, mean treatment duration 5.1 years, and 47 untreated MS patients) and a longitudinal study (33 patients with MS before and during NTZ, mean treatment duration: 4.8 years), analyzing paired serum and CSF samples for IgG, IgA, and IgM levels, reactivity against selected viruses (measles virus, rubella virus, and varicella zoster virus [MRZ] reaction), and oligoclonal bands (OCBs). Banding patterns before and after therapy were directly compared by isoelectric focusing in 1 patient. In addition, we determined the expression of α4-integrin by FACS analysis on blood-derived B-cell subsets (plasmablasts, memory B cells, and naive B cells) of healthy controls.ResultsIn serum, NTZ decreased IgM and IgG, but not IgA, levels. IgM hypogammaglobulinemia occurred in 28% of NTZ-treated patients. In CSF, NTZ treatment resulted in a strong reduction of intrathecally produced IgG and, to a lesser extent, IgA, whereas IgM indices [(Ig CSF/Serum)/(Albumin CSF/Serum)] remained largely unchanged. Reduction of the IgG index correlated with NTZ treatment duration, as did serum IgM and IgA levels. MRZ reaction was unchanged and OCB persisted. Direct comparison of OCB pattern before and after NTZ revealed the persistence of individual bands. α4-Integrin expression was highest on plasmablasts (CD19+CD38+CD27+).ConclusionOur data indicate that NTZ reduces short-lived plasmablasts in the CNS compartment but has little effect on locally persisting long-lived plasma cells.

Temporary rivers become dependent on wastewater effluent for base flows, which severely impacts river ecosystems through exposure to elevated levels of nutrients, dissolved organic matter, and organic micropollutants. However, biodegradation processes occurring in these rivers can be enhanced by wastewater bacteria/biofilms. Here, we evaluated the attenuation of pharmaceuticals and their human metabolites performing retrospective analysis of 120 compounds (drugs, their metabolites and transformation products) in mesocosm channels loaded with wastewater effluents twice a week for a period of 31 days. Eighteen human metabolites and seven biotransformation products were identified with high level of confidence. Compounds were classified into five categories. Type-A: recalcitrant drugs and metabolites (diclofenac, carbamazepine and venlafaxine); Type-B: degradable drugs forming transformation products (TPs) (atenolol, sitagliptin, and valsartan); Type-C: drugs for which no known human metabolites or TPs were detected (atorvastatin, azithromycin, citalopram, clarithromycin, diltiazem, eprosartan, fluconazole, ketoprofen, lamotrigine, lormetazepam, metformin, telmisartan, and trimethoprim); Type-D: recalcitrant drug metabolites (4-hydroxy omeprazole sulfide, erythro/threo-hydrobupropion, and zolpidem carboxylic acid); Type-E: unstable metabolites whose parent drug was not detectable (norcocaine, benzolylecgonine, and erythromycin A enol ether). Noteworthy was the valsartan acid formation from valsartan with transient formation of TP-336., This study has been financially supported by the EU through the PRIMA project (INWAT 201980E121). This work was supported by the Spanish Ministry of Science and Innovation (Project CEX2018-000794-S). The authors also acknowledge SCIEX for providing the loan instrument LC/HRMS QTOF X500R. The EU is not liable for any use that may be made of the information contained therein.

While some COVID-19 patients maintain SARS-CoV-2-specific serum IgGs for more than 6 months post-infection, others, especially mild cases, eventually lose IgG levels. We aimed to assess the persistence of SARS-CoV-2-specific B cells in patients who have lost specific IgGs and analyzed the reactivity of the immunoglobulins produced by these B cells. Circulating IgG memory B cells specific for SARS-CoV-2 were detected in all 16 patients 1–8 months post-infection, and 11 participants had specific IgA B cells. Four patients lost specific serum IgG after 5–8 months but had SARS-CoV-2-specific-B-cell levels comparable to those of seropositive donors. Immunoglobulins produced after in vitro differentiation blocked receptor-binding domain (RBD) binding to the cellular receptor ACE-2, indicating neutralizing activity. Memory-B-cell-derived IgGs recognized the RBD of B.1.1.7 similarly to the wild-type, while reactivity to B.1.351 and P.1. decreased by 30% and 50%, respectively. Memory-B-cell differentiation into antibody-producing cells is a more sensitive method for detecting previous infection than measuring serum antibodies. Circulating SARS-CoV-2 IgG memory B cells persist, even in the absence of specific serum IgG; produce neutralizing antibodies; and show differential cross-reactivity to emerging variants of concern. These features of SARS-CoV-2-specific memory B cells will help to understand and promote long-term protection. Funding: This work was supported by the DFG (SFB TR128) and the MOMENTE program LMU (to SM). Declaration of Interest: None to declare.

Objectives During the COVID-19 pandemic, SARS-CoV-2 antibody testing has been suggested for (1) screening populations for disease prevalence, (2) diagnostics, and (3) guiding therapeutic applications. Here, we conducted a detailed clinical evaluation of four Anti-SARS-CoV-2 immunoassays in samples from acutely ill COVID-19 patients and in two negative cohorts. Methods 443 serum specimens from serial sampling of 29 COVID-19 patients were used to determine clinical sensitivities. Patients were stratified for the presence of acute respiratory distress syndrome (ARDS). Individual serum specimens from a pre-COVID-19 cohort of 238 healthy subjects and from a PCR-negative clinical cohort of 257 patients were used to determine clinical specificities. All samples were measured side-by-side with the Anti-SARS-CoV-2-ELISA (IgG), Anti-SARS-CoV-2-ELISA (IgA) and Anti-SARS-CoV-2-NCP-ELISA (IgG) (Euroimmun AG, Lübeck, Germany) and the Elecsys Anti-SARS-CoV-2 ECLIA (Roche Diagnostics International, Rotkreuz, Switzerland). Results Median seroconversion occurred earlier in ARDS patients (8–9 days) than in non-ARDS patients (11–17 days), except for EUR N-IgG. Rates of positivity and mean signal ratios in the ARDS group were significantly higher than in the non-ARDS group. Sensitivities between the four tested immunoassays were equivalent. In the set of negative samples, the specificity of the Anti-SARS-CoV-2-ELISA (IgA) was lower (93.9%) compared to all other assays (≥98.8%) and the specificity of Anti-SARS-CoV-2-NCP-ELISA (IgG) was lower (98.8%) than that of Elecsys Anti-SARS-CoV-2 (100%). Conclusions Serial sampling in COVID-19 patients revealed earlier seroconversion and higher signal ratios of SARS-CoV-2 antibodies as a potential risk marker for the development of ARDS, suggesting a utility for antibody testing in acutely diseased patients.

SummaryAntibody-mediated encephalitis has been discussed as one possible cause for isolated psychotic syndromes. Mostly based on serum samples, findings have been controversial. We present the results of a retrospective study of 124 clinically diagnosed psychotic patients without documented relevant neurological symptoms. All were tested for different antineuronal antibodies in cerebrospinal fluid (CSF) while 81 received serum testing. Antineuronal antibodies in CSF were negative across the sample. 3.7% showed low positive serum antibodies. Our findings highlight the importance of a deeper discussion about the relevance of low positive serum antibodies without concurrent findings in CSF or clinical signs for autoimmune encephalitis.Declaration of interestNone.

Myositis-associated antibodies (MAA) and myositis-specific antibodies (MSA) are detected in patients with idiopathic inflammatory myopathies (IIM); their role as diagnostic biomarkers is however still debated. The aim of our study was to assess the utility of MAA/MSA assessed by new line immunoassays in detecting myositis among neuromuscular patients. We retrospectively analysed sera samples obtained from patients tested for myositis antibodies with the “Euroline: Autoimmune Inflammatory Myopathies 16Ag” and “myositis profile 3” kits (Mi-2, TIF1γ, MDA5, NXP2, SAE1, Jo-1, SRP, PL-7/12, EJ, OJ, Ro-52, Ku, PM-Scl75/100). First symptom, CK, EMG, muscle biopsy and diagnosis were also analysed. Using logistic regression analysis, two diagnostic models were built to evaluate the diagnostic power of MAA/MSA in distinguishing myositis patients from controls and other myopathies. 1229 patients were identified. 141 patients had a bioptic confirmed IIM; other diagnoses included: myopathy (n = 357), other neuromuscular diseases (n = 144) and no neuromuscular diseases (n = 587). The specificity was 95% for MSA and 89% for MAA, the sensitivity 20% and 22%, respectively. MAA showed no use in differentiating myositis patients from controls, whereas MSA had limited effect (OR = 5.165), compared to other variables as EMG (OR = 47.755) or CK > 2000 U/L (OR = 45.307). MSA were, however, the most useful parameter differentiating IIM from non-IIM patients (OR = 7.259), better than CK > 2000 U/L (OR = 4.033) and MAA (OR = 2.737). Line immunoassays for myositis antibodies show high specificity but low sensitivity. Their usefulness as diagnostic biomarkers widely depends on the clinical settings. Our study suggests that MSA/MAA should be used for confirmatory and differential diagnosis rather than for screening purposes in inflammatory myopathies.

Background CSF abnormalities and a neuroinflammatory pathophysiology have been discussed for affective and non-affective psychosis for more than 30-years. Recent studies pointed towards a specific phenotype of autoimmune-antibody mediated psychosis, but evidence is still sparse. Especially CSF data investigating autoimmune antibodies in large-scale CSF cohorts of affective and non-affective psychoses are lacking. Methods We analyzed a retrospective naturalistic cohort of 592 patients with A) schizophrenia-spectrum disorders (N = 330) or B) depressive disorders (N = 262) who underwent a lumbar puncture as part of the clinical routine in the Department of Psychiatry and Psychotherapy at the Ludwig-Maximilians University Munich between July 2012 and May 2017. We used a predefined systematic algorithm for the database search in the clinical documentation system and data was extracted by TO and AG. The study was approved by the local ethics committee. Results We identified 592 patients with standard CSF parameters. Schizophrenia spectrum patients did not differ from depressive patients with regard to the white blood cell count (cells/µl) (p = 0.774) or albumin quotient (p = 0.663). The general prevalence of oligoclonal bands did not differ between groups (schizophrenia: 37.0%, depression: 37.8%; p = 0.838). However, schizophrenia patients showed higher frequencies for intrathecal oligoclonal bands (32% of all oligoclonal bands) compared to depressive patients (19.1% of all oligoclonal bands. (p = 0.034). 124 schizophrenia-spectrum patients (54 first-episode patients) received CSF analyses for neural antibodies. None of the patients showed positive CSF results in any of the tested autoimmune-encephalitis panel (NMDA(N=119), AMPA-1(N=114), AMPA-2(N=114), CASPR(N=111), LGI-1(N=110) and GABA-B(N=112)-Antibodies) in CSF. The results for the intracellular onconeuronal and synaptic antibodies were also negative (Amphyphysin(N=93), Yo(N=58), Hu(N=94), Ri(N=94), CV2(N=93), Ma1(N=93) and Ma2(N=93)-Antibodies). Three of these patients with negative CSF titers did have low-titer neuronal antibodies in serum: CASPR-2-AB: 1:10, CASPR-2-AB: 1:50, Yo-AB: low band-intensity. 36 depression patients were also tested for autoimmune antibodies and again no positive reports could be identified in CSF. Discussion This is the first analyses of autoimmune antibodies in first-episode and recurrent schizophrenia and depressive mood disorder showing no positive CSF titers. However, schizophrenia patients have a higher prevalence of intrathecal oligoclonal bands compared to affective patients pointing towards more immunological disturbances in this population. The here presented analyses are exploratory and need to undergo confirmatory analyses and quality control.

Spleen tyrosine kinase (Syk) is essential for signal transduction of immunoreceptors. Inhibition of Syk abrogates mast cell degranulation and B cell responses. We hypothesized that Syk inhibition in the lung by inhaled route could block airway mast cells degranulation and the early asthmatic response without the need of systemic exposure. We discovered LAS189386, a novel Syk inhibitor with suitable properties for inhaled administration. The aim of this study was to characterize the in vitro and in vivo profile of LAS189386. The compound was profiled in Syk enzymatic assay, against a panel of selected kinases and in Syk-dependent cellular assays in mast cells and B cells. Pharmacokinetics and in vivo efficacy was assessed by intratracheal route. Airway resistance and mast cell degranulation after OVA challenge was evaluated in an ovalbumin-sensitized Brown Norway rat model. LAS189386 potently inhibits Syk enzymatic activity (IC50 7.2 nM), Syk phosphorylation (IC50 41 nM), LAD2 cells degranulation (IC50 56 nM), and B cell activation (IC50 22 nM). LAS189386 inhibits early asthmatic response and airway mast cell degranulation without affecting systemic mast cells. The present results support the hypothesis that topical inhibition of Syk in the lung, without systemic exposure, is sufficient to inhibit EAR in rats. Syk inhibition by inhaled route constitutes a promising therapeutic option for asthma.

10 pages, 5 figures, 1 table, supplementary material https://dx.doi.org/10.1007/s00216-017-0467-7, The widespread occurrence of pharmaceuticals in the aquatic environment has raised concerns about potential adverse effects on exposed wildlife. Very little is currently known on exposure levels and clearance mechanisms of drugs in marine fish. Within this context, our research was focused on the identification of main metabolic reactions, generated metabolites, and caused effects after exposure of fish to carbamazepine (CBZ) and ibuprofen (IBU). To this end, juveniles of Solea senegalensis acclimated to two temperature regimes of 15 and 20 °C for 60 days received a single intraperitoneal dose of these drugs. A control group was administered the vehicle (sunflower oil). Bile samples were analyzed by ultra-high-performance liquid chromatography–high-resolution mass spectrometry on a Q Exactive (Orbitrap) system, allowing to propose plausible identities for 11 metabolites of CBZ and 13 metabolites of IBU in fish bile. In case of CBZ metabolites originated from aromatic and benzylic hydroxylation, epoxidation, and ensuing O-glucuronidation, O-methylation of a catechol-like metabolite was also postulated. Ibuprofen, in turn, formed multiple hydroxyl metabolites, O-glucuronides, and (hydroxyl)-acyl glucuronides, in addition to several taurine conjugates. Enzymatic responses after drug exposures revealed a water temperature-dependent induction of microsomal carboxylesterases. The metabolite profiling in fish bile provides an important tool for pharmaceutical exposure assessment, This study has been financially supported by the EU through the FP7 project GLOBAQUA (Grant agreement no. 603629), partly supported by the MEC [NET-SCARCE (CTM2015-69780-REDC) and by the Generalitat de Catalunya (Consolidated Research Group “2014 SGR 418—Water and Soil Quality Unit”). This work is also integrated in the MINECO project CTM 2010-16611, DEPURAMAR

Background Autoantibodies, in particular those against aquaporin-4 and myelin-oligodendrocyte glycoprotein (MOG), aid as biomarkers in the differential diagnosis of demyelination. Here, we report on discovery of autoantibodies against flotillin in patients with multiple sclerosis (MS). Methods The target antigen was identified by histo-immunoprecipitation using the patients’ sera and cryosections of rat or pig cerebellum combined with mass spectrometrical analysis. Correct identification was ascertained by indirect immunofluorescence and neutralization tests using the target antigens recombinantly expressed in HEK293 cells. Results Serum and CSF of the index patient produced a fine-granular IgG indirect immunofluorescence staining of the hippocampal and cerebellar molecular layers. Flotillin-1 and flotillin-2 were identified as target autoantigens. They also reacted with recombinant human flotillin-1/2 co-expressed in HEK293 cells, but not with the individual flotillins in fixed- and live-cell assays. Moreover, neutralization using flotillin-1/2, but not the single flotillins, abolished the tissue reactivity of patient serum. Screening of 521 patients, for whom anti-aquaporin-4 testing was requested and negative, revealed 8 additional patients with anti-flotillin-1/2 autoantibodies. All eight were negative for anti-MOG. Six patients ex post fulfilled the revised McDonald criteria for MS. Vice versa, screening of 538 MS sera revealed anti-flotillin-1/2 autoantibodies in eight patients. The autoantibodies were not found in a cohort of 67 patients with other neural autoantibody-associated syndromes and in 444 healthy blood donors. Conclusions Autoantibodies against the flotillin-1/2 heterocomplex, a peripheral membrane protein that is involved in axon outgrowth and regeneration of the optic nerve, are present in 1–2% of patients with bona fide MS. Electronic supplementary material The online version of this article (doi:10.1186/s12974-017-0900-z) contains supplementary material, which is available to authorized users.

Pruritus is a major symptom of several dermatological diseases but has limited therapeutic options available. Animal models replicating the pathophysiology of pruritus are needed to support the development of new drugs. Induction of pruritus by chloroquine (CQ) in mice is widely used, although, as with similar models, it has low throughput and does not distinguish between antipruritic effects and confounding factors such as sedation. To overcome these issues, we incorporated into the model an automated system that measures both scratching and locomotor behaviour simultaneously. We combined this system with the determination of CQ levels in different tissues to understand the impact of the route of CQ administration on the pruritogenic response. We concluded that whereas oral CQ does not induce pruritus due to insufficient skin levels, the bell-shaped curve of pruritus observed following subcutaneous administration is due to toxicity at high doses. We validated the model with several drugs currently used in humans: nalfurafine, aprepitant, cyproheptadine and amitriptyline. By comparing the effects of the drugs on both scratching and locomotor activity, we concluded that nalfurafine and aprepitant can exhibit efficacy at doses devoid of central effects, whereas central effects drove the efficacy of the other two drugs. This was further confirmed using non-brain-penetrant drugs. Moreover, as anticipated, anti-inflammatory drugs showed no efficacy. In conclusion, the use of an automated integrated behavioural assessment in CQ-induced pruritus makes the assay suitable for screening purposes and allows for a correct interpretation of the antipruritic effect of the compounds evaluated.

BACKGROUND: A polyspecific, intrathecal humoral immune response against neurotropic viruses such as measles, rubella and varicella zoster virus (MRZ reaction, MRZR) is present in 80-100% of patients with multiple sclerosis (MS), but has not to date been evaluated in patients with neuromyelitis optica (NMO). AIMS: To evaluate whether MRZR distinguishes NMO and MS. METHODS: 20 patients with NMO and 42 with MS were included. The intrathecal synthesis of antibodies against measles, rubella and varicella zoster virus was detected by calculation of the respective antibody indices (AI). RESULTS: A positive MRZ reaction, as defined by a combination of at least two positive AIs, was found in 37/42 MS, but in only 1/20 NMO patients (p

The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTh2) is a G protein-coupled receptor expressed on the leukocytes most closely associated with asthma and allergy like eosinophils, mast cells, Th2-lymphocytes and basophils. At present it is clear that CRTh2 mediates most prostaglandin D2 (PGD2) pro-inflammatory effects and as a result antagonists for this receptor have reached asthma clinical studies showing a trend of lung function improvement. The challenge remains to identify compounds with improved clinical efficacy when administered once a day. Herein we described the pharmacological profile of LAS191859, a novel, potent and selective CRTh2 antagonist. In vitro evidence in GTPγS binding studies indicate that LAS191859 is a CRTh2 antagonist with activity in the low nanomolar range. This potency is also maintained in cellular assays performed with human eosinophils and whole blood. The main differentiation of LAS191859 vs other CRTh2 antagonists is in its receptor binding kinetics. LAS191859 has a residence time half-life of 21h at CRTh2 that translates into a long-lasting in vivo efficacy that is independent of plasma levels. We believe that the strategy behind this compound will allow optimal efficacy and posology for chronic asthma treatment.

Recent publications on pharmaceutical monitoring are increasingly covering the field of illicit drugs and lately the forensic evaluation of designing illegal analogs of lifestyle drugs like the phosphodiesterase type 5 (PDE-5) inhibitors Viagra (sildenafil), Levitra (vardenafil) and Cialis (tadalafil). Recently, the presence of all three erectile dysfunction treatment drugs has been reported in wastewaters at very low concentrations. In the environment, contaminants undergo various physical or chemical processes classified into abiotic (photolysis, hydrolysis) and biotic (biodegradation) reactions. Thus, changes in the chemical structure lead to the formation of new transformation products, which may persist in the environment or be further degraded. This study describes the photolysis of sildenafil (SDF) and its human metabolite N-demethylsildenafil (DM-SDF) under simulated solar radiation (Xenon lamp). Following chromatographic separation of the irradiated samples, eight photoproducts in the SDF samples and six photoproducts for DM-SDF were detected and characterized. The combination of ultra performance liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (UPLC-ESI-QToF-MS), liquid chromatography-atmospheric pressure chemical ionization-triple quadrupole mass spectrometry (LC-APCI-QqQ-MS) and hydrogen/deuterium-exchange experiments allowed to propose plausible chemical structures for the photoproducts, taking into account the characteristic fragmentation patterns and the accurate mass measurements. These mass spectral data provided sound evidence for the susceptibility of the piperazine ring toward photodegradation. A gradual breakdown of this heterocyclic structure gave rise to a series of products, which in part were identical for SDF and DM-SDF. The sulfonic acid, as the formal product of sulfonamide hydrolysis, was identified as key intermediate in the photolysis pathway. In both drug/metabolite molecules, phototransformation processes taking place beyond the sulfonamide group were deemed to be of minor relevance.

Primary biliary cirrhosis, primary sclerosing cholangitis and autoimmune hepatitis are the three major immune-mediated liver diseases. The etiologies of primary biliary cirrhosis, primary sclerosing cholangitis and autoimmune hepatitis are largely unknown, but seem to be influenced by genetic and environmental factors. Autoantibodies can be found in nearly all patients with primary sclerosing cholangitis and autoimmune hepatitis, and in the vast majority of patients with primary sclerosing cholangitis. In addition, autoimmune hepatitis is associated with high concentrations of serum globulins. Enhanced liver cell death by apoptosis has been described in all of these liver diseases, although the precise mechanisms remain unclear. In general, apoptosis can be initiated via an extrinsic pathway that is triggered by engagement of death receptors on the cell surface, or via an intrinsic pathway that is induced by mitochondrial injury and is influenced by members of the Bcl-2 family. In both pathways, effector caspases are finally activated that cleave and degrade cell structures, resulting in the release of apoptotic products into the circulation. New diagnostic tests can detect these apoptotic markers and programmed cell death ligands such as Fas and Fas-ligands, nucleosomes, caspases, cytokeratin fragments, macrophage migration inhibitory factor, soluble intracellular adhesion molecule, natural killer cells group 2D and programmed death ligands. Several of these markers have been found to be altered in tissue and/or blood of immune-mediated liver diseases, some also in nonimmune-mediated liver diseases. Beyond their potential usefulness as additional diagnostic markers, they may be valuable for the estimation of disease severity and therapy monitoring. This review summarizes current knowledge on apoptotic mechanisms, death receptor ligands and circulating apoptotic markers in immune-mediated liver diseases.

Introduction: Treatment of chronic cough is an unmet medical need impairing patients9 quality of life. Transient Receptor Potential Ankyrin 1 (TRPA1) channels may provide a safe opportunity to target cough response at the periphery of the cough reflex arc. Poor potency and physicochemical properties of available TRPA1 antagonists are hampering their advance in the clinics. Here we describe a novel, potent and orally available TRPA1 antagonist, compound A, and its profile in preclinical models of cough. Objectives To profile the TRPA1 antagonist Compound A in in vitro assays and in a preclinical model of induced cough. In vitro profile was compared with that of HC030031. Methods: Potency of the compounds was assessed in calcium fluorescence based assays and electrophysiology in cells overexpressing TRPA1. Inhibition of isolated guinea pig (gp) vagus nerve depolarization induced by acrolein was used as functional in vitro assay. Cough was induced upon nebulization of 10mM Allyl Isothiocyanate (AITC) for 10 minutes in conscious gp individually placed into whole body plethysmography chambers (Buxco). Results: Compound A showed a potent TRPA1 antagonist activity with enhanced efficacy at inhibiting isolated gp vagus nerve depolarization (EC50=80nM). Favorable oral pharmacokinetic profile translated in a relevant efficacy at inhibiting cough induced by AITC in gp (ED50=0.17mg/kg). Conclusions: Compound A is an orally available, potent and novel TRPA1 antagonist that has proven to be efficacious in a mechanistic model of induced cough. Promising profile of Compound A suggests it could be useful for the treatment of chronic cough associated to respiratory diseases.

A study was conducted to examine the influence of hydraulic retention time (HRT) and solid retention time (SRT) on the removal of tetracycline in the activated sludge processes. Two lab-scale sequencing batch reactors (SBRs) were operated to simulate the activated sludge process. One SBR was spiked with 250 microg/L tetracycline, while the other SBR was evaluated at tetracycline concentrations found in the influent of the wastewater treatment plant (WWTP) where the activated sludge was obtained. The concentrations of tetracyclines in the influent of the WWTP ranged from 0.1 to 0.6 microg/L. Three different operating conditions were applied during the study (phase 1-HRT: 24 h and SRT: 10 days; phase 2-HRT: 7.4 h and SRT: 10 days; and phase 3-HRT: 7.4 h and SRT: 3 days). The removal efficiency of tetracycline in phase 3 (78.4 +/- 7.1%) was significantly lower than that observed in phase 1 (86.4 +/- 8.7%) and phase 2 (85.1 +/- 5.4%) at the 95% confidence level. The reduction of SRT in phase 3 while maintaining a constant HRT decreased tetracycline removal efficiency. Sorption kinetics reached equilibrium within 24 h. Batch equilibrium experiments yielded an adsorption coefficient (Kads) of 8400 +/- 500 mL/g and a desorption coefficient (Kdes) of 22 600 +/- 2200 mL/g. No evidence of biodegradation for tetracycline was observed during the biodegradability test, and sorption was found to be the principal removal mechanism of tetracycline in activated sludge.

Effects of alpha-melanocyte-stimulating hormone (alpha-MSH), beta-melanocyte-stimulating hormone (beta-MSH), beta-lipotropin (beta-LPH), and beta-endorphin (beta-EPH) at concentrations from 10(-9) M up to 10(-6) M on human adipose tissue lipoprotein lipase (LPL) were studied in a cell-free system. alpha-MSH and beta-MSH did not exert any effect on LPL; no degradation of these peptides in the incubation medium could be detected by HPLC analysis. beta-LPH and beta-EPH failed to alter enzyme activity. However, HPLC analysis revealed an unspecific rapid degradation of the peptides due to the activity of tissue proteases. Therefore, the protease inhibitors amastatin, antipain, APMSF, and TPCK were tested at concentrations of 10(-5), 10(-4), and 10(-3) M for their efficacy to inhibit degradation. None of the inhibitors was able to substantially reduce proteolysis of beta-LPH, as was the case with amastatin, APMSF, and TPCK for beta-EPH. However, antipain at 10(-4) M preserved at least 20% of the initial peptide concentration from proteolysis up to 150 min. Antipain caused a decrease in lipoprotein lipase activity (LPLA), which was dependent on concentration. The adverse effect of antipain at concentrations of 10(-4) M on LPL was completely abolished by beta-EPH at a concentration of 10(-6) M.

Objectives: Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. Epidemiological work traced the origin to the ingestion of aniline-adulterated rapeseed oil, fraudulently marketed and sold as edible oil. It affected more than 20,000 people with over 400 deaths in the first 2 years. In 2001 evidence was presented that genetic factors could play a role in the susceptibility of individuals to the disease. Thus, a prospective study on the differences in gene expression in sera between control versus TOS-affected populations, both originally exposed to the toxic oil, was undertaken in our laboratory. Methods: Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Problems related with serum analysis by 2-DE were addressed to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins. The use of nonionic reductants or the presence of thiourea in the gels, were also tested. Results: From the resulting optimized images, a group of 329 major gel spots was located, matched and compared with serum samples. Thirty-five of these protein spots were found to be under- or over-expressed in TOS patients (threefold increase or decrease). Proteins in these spots were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) peptide map fingerprinting and database search. Several haptoglobin (Hp) isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS. Resolution of the homologous α-1s and α-1f chains, with a mass difference of only 0.043 Da, was obtained after guanidation of the protein with O-methylisourea. We applied this procedure to the study of the distribution of the Hp alleles HP 2, HP 1s and HP 1f in control versus TOS-affected populations. The MALDI-TOF proteotyping method was validated by a parallel analysis of the serum samples by 2-DE. Conclusions: Data obtained from 54 TOS cases and 48 controls indicate significant differences in the distribution of Hp phenotypes in the two populations. Haptoglobin phenotypes have been reported to have biological and clinical consequences and have been described as risk factors for several diseases. Consequently, it was concluded that haptoglobin polymorphism could play a role in TOS. © 2010 Elsevier Ireland Ltd. All Rights Reserved., This work was supported by WHO projects EU/03/016989 and EU 05/025039.

This article describes the photolysis of azithromycin, a macrolide antibiotic with reported occurrence in environmental waters, under simulated solar radiation. The photodegradation followed first-order reaction kinetics in five matrices examined. In HPLC water, the degradation rate was the slowest (half-life: 20 h), whereas in artificial freshwater supplemented with nitrate (5 mg L−1) or humic acids (0.5 mg L−1) the degradation of azithromycin was enhanced by factors of 5 and 16, respectively, which indicated the role of indirect photolysis involving the formation of highly reactive species. Following chromatographic separation on a UPLC system, the characterization of the transformation products was accomplished using high-resolution QqToF-MS analysis. The presence of seven photoproducts was observed and their formation was postulated to originate from (bis)-N-demethylation in the desosamine sugar, O-demethylation in the cladinose sugar, combinations thereof, as well as from hydrolytic cleavages of the desosamine and/or cladinose residue. Two of these photoproducts could also be detected in natural photodegradation process in river water which was spiked with azithromycin.

The highly polar, nonionic X-ray contrast agent iopromide (C(18)H(24)N(3)O(8)I(3); 791 Da) is resistant to microbial degradation during the activated sludge process in wastewater treatment plants and hence is released into the aquatic environment. Against this background, the present study was conducted to evaluate the phototransformation, potentially constituting the most relevant removal mechanism in rivers and streams. The photolysis of the iodinated aromatic compound was investigated in a Suntest solar simulator using aqueous solutions. Following a 120-min irradiation period, an almost complete primary degradation of iopromide gave rise to a series of photoproducts that were chromatographed on a reversed-phase UPLC and subsequently characterized by a combination of accurate mass measurements on a ESI-QqToF-MS instrument and H/D-exchange experiments. This analytical approach facilitated confident identification of eight prominent products with the following elemental compositions and molecular weights: C(18)H(25)N(3)O(9)I(2) (681 Da); C(18)H(25)N(3)O(8)I(2) (665 Da); C(17)H(23)N(3)O(8)I(2) (651 Da); C(18)H(24)N(3)O(9)I (553 Da); C(17)H(24)N(3)O(8)I (525 Da); C(15)H(20)N(3)O(6)I (465 Da); C(14)H(18)N(3)O(6)I (451 Da); and C(18)H(25)N(3)O(9) (427 Da). Their formation was the result of four principal photoreactions: (1) gradual, and eventually complete, deiodination of the aromatic ring; (2) substitution of the halogen by a hydroxyl group; (3) N-dealkylation of the amide in the hydroxylated side chain; and (4) oxidation of a methylene group in the hydroxylated side chain to the corresponding ketone. In conclusion, the findings of the artificial sunlight irradiation experiments indicated that in real environmental settings iopromide might suffer partial or even complete deiodination.

Background 80–100% of patients with multiple sclerosis (MS) display a polyspecific, intrathecal humoral immune response against a broad panel of viral agents including antibodies to measles, rubella and varicella zoster virus as its three most abundant components (called MRZ reaction [MRZR]). However, a positive MRZR reaction can also be found in some patients with CNS vasculitis, another rare autoimmune condition, raising the question whether this marker is really of high specificity for MS as previously claimed or whether it just represents a non-specific marker of CNS autoimmunity. Besides MS and CNS vasculitis, paraneoplastic neurological disorders (PND) represent the best recognized models of CNS autoimmunity. Objective To investigate MRZR for the first time in patients with PND. Patients and methods Forty-two patients with MS and 34 with PND were compared in this study. The intrathecal synthesis of antibodies against measles, rubella, and varicella zoster virus was detected by calculation of the respective antibody indices (AI). Results A positive MRZ reaction as defined by a combination of at least two positive AIs was present in 37/42 patients with MS, but in none of the patients with PND ( p p Conclusions Our results confirm that MRZR is not a general marker of CNS autoimmunity. Taking into account the very rarity of CNS vasculitis as well the lack of MRZR positivity in infectious inflammatory CNS conditions as previously demonstrated, MRZR might indeed be a promising marker of MS. Further investigations on MRZR in more rare autoimmune conditions, which were not available for analysis in this study, are now warranted to refine further the specificity of this parameter.

Intravenous immunoglobulin (IVIg) preparations are increasingly used for therapy of several neuroimmunologic diseases. IVIg therapy is considered safe, although serious side effects like aseptic meningitis, cerebral vasospasm, or ischemic encephalopathy have been reported. These side effects are frequently associated with neutrophilic pleocytosis in the cerebrospinal fluid (CSF), suggesting a neutrophil-mediated mechanism. To elucidate the potential role of neutrophil activation, we analyzed IVIg preparations from 5 different commercial sources for the presence of antineutrophil cytoplasmic antibody (ANCA)–like immunoglobulins against ethanol-fixed peripheral-blood neutrophils, purified human antigens, and a panel of human and nonhuman tissues. All IVIg batches tested (n = 13) contained atypical ANCAs (IgG titer up to 1:2048, IgA up to 1:512). Moreover, all preparations were capable of inducing hydrogen peroxide production in TNFα-primed human neutrophils, with a significant correlation (P < .005) between atypical ANCA titers in IVIg preparations and neutrophil activation. Fc-mediated binding and activation was ruled out by the use of IVIg-F(ab′)2 fragments. Our findings strongly suggest that in vivo activation of TNFα-primed neutrophils by atypical ANCAs of IVIg may contribute to the side effects of IVIg therapy and for the first time demonstrate that the activation of neutrophil granulocytes by IVIg occurs in an Fc receptor (FcR)–independent, hence antigen-dependent, way.

8 pages, 2 tables, 5 figures.-- PMID: 17914752 [PubMed].-- Printed version published Nov 1, 2007., Supplementary information (PDF file, 2 pages, 1 fig, Word file, 2 pages, 1 fig) available at: http://pubs.acs.org/doi/suppl/10.1021/ac070891u, In the environment, organic micropollutants such as pharmaceuticals can be degraded via various biotic and abiotic transformation routes. In surface waters, for example, photodegradation may constitute a relevant natural attenuation process for drug residues that have been discharged from sewage treatment facilities. In the present work, the photochemical fate of the prodrug enalapril (376 Da, C(20)H(28)N2O5) and its active metabolite enalaprilat (348 Da, C(18)H(24)N2O5), a hypotensive cardioprotector previously reported to occur in contaminated rivers, was investigated in aqueous media under the influence of irradiation generated by a sunlight simulator. The experiments yielded three detectable photodegradates for enalapril (346 Da, 2 x 207 Da) whereas the photolysis of enalaprilat went hand in hand with the intermittent buildup of one photodegradate (304 Da). Fragmentation patterns of the parent compounds were established on a hybrid quadrupole-linear ion trap-mass spectrometer exploiting its MS3 capabilities. Accurate mass measurements recorded on a hybrid quadrupole-time-of-flight instrument in MS/MS mode allowed us to propose elemental compositions for the molecular ions of the degradates (346 Da, C(19)H(26)N2O4; 207 Da, C(12)H(17)NO2; 304 Da, C(17)H(24)N2O3) as well as of their fragment ions. Based on these complementary data sets from the two distinct mass spectrometric instruments, plausible structures were postulated for the four photodegradates. The compounds formed by enalapril corresponded to the loss of formaldehyde out of the proline residue (346 Da), cleavage of the central amide bond (207 Da) followed by migration of the ethylester side chain (207 Da) while decarboxylation of the free carboxylic acid was described for enalaprilat (304 Da). The study emphasized the potential of sunlight for breaking down an environmentally relevant drug and its metabolite., The work presented in this article was supported by the EU Project EMCO-INCO-CT-2004-509188) and by the Spanish Ministerio de Educación y Ciencia, Project EVITA (CTM2004-06255-CO3-01). This work reflects only the author’s views and the European Community is not liable for any use that may be made of the information contained therein. S.P. acknowledges a postdoctoral contract from I3P Program (Itinerario Integrado de Inserción Profesional); co-financed by CSIC and European Social Funds. S.P. acknowledges Roser Charler and Dori Fanjul, for their support with the MS instrumentation.

The inadequate response of immune cells to circulating apoptotic products, such as nucleosomal DNA fragments, is assumed to be a potent stimulus for the production of autoantibodies during the pathogenesis and progression of systemic lupus erythematosus (SLE). Here, we analyzed the levels of circulating nucleosomes, caspases, and C-reactive protein in sera of 244 individuals with various autoimmune diseases (155 with autoimmune hepatic disorders, 25 with ANCA-associated vasculitis, and 64 with various connective tissue diseases), and 32 healthy controls. Nucleosomes and caspase activities were significantly elevated in sera of patients with hepatic autoimmune diseases, connective tissue diseases, and particularly in ANCA-associated vasculitis when compared with healthy individuals. Nucleosomes showed a correlation with caspases, and caspases with C-reactive protein, but nucleosomes did not correlate with C-reactive protein. Serum levels of the apoptotic products, nucleosomes, and caspases are increased in various autoimmune diseases but may not be solely responsible for antinucleosome antibody production in SLE patients. It remains to be clarified whether qualitative changes in nucleosomes are linked with pathogenesis and disease progression in SLE.

Antineuronal antibody-associated paraneoplastic neurological syndromes (PaNSs) result from tumour-stimulated autoimmune attacks against components of the nervous system. The rare antineuronal antibody anti-Ri (ANNA-2) was initially thought to be closely associated with paraneoplastic opsoclonus–myoclonus syndrome. Recently, however, it has been found in several other PaNSs.1 First-line treatment in PaNSs is removal of the underlying tumour. Second-line treatment is immunosuppression, which, although extensively used, especially when no tumour is detected, is often ineffective. Often, patients die from relentlessly progressive PaNS rather than the underlying neoplasm.2 Here, we report the 2-year follow-up of an anti-Ri-positive steroid-responsive myeloneuropathy. No tumour was detected. Immunosuppressive treatment was tailored on the basis of clinical relapses, inflammatory changes in cerebrospinal fluid (CSF) and somatosensory evoked potentials (SEPs). A 65-year-old woman, a retired administrator, was referred to our department with a 10-month history of progressive gait difficulties and ascending sensory loss in her legs, eventually having become wheelchair bound. Her medical history was unremarkable apart from enlarged axillary lymph nodes that were excised 9 months earlier. Histological examination showed only inflammatory changes. She had received hormone replacement therapy for 11 years. She never smoked. On neurological examination, deep tendon reflexes were absent. She had spastic paraparesis with bilateral extensor plantar responses. Bilateral hypaesthesia for light touch up to the knees and diminished vibration sense were observed distally in her legs. Magnetic resonance imaging (MRI) showed symmetrical T2-hyperintense multisegmental (C6–TH3/TH8–TH12) cervicothoracal lesions of the spinal cord, with gadolinium enhancement restricted to the lateral parts (fig 1A). Cerebral …

9 pages, 9 figures.-- PMID: 16536422 [PubMed].-- Printed version published Mar 15, 2006., Identification of degradation products of environmental contaminants is a challenging task because not only are they present in very low concentrations but they are also mixed with complex matrixes that interfere with detection. This work illustrates a simple approach using ion trap mass spectrometry combined with H/D-exchange experiments to elucidate the structures of iopromide metabolites formed during biodegradation in activated sludge. Iopromide is an X-ray contrast agent that has been detected frequently in effluents of wastewater treatment plants and in surface waters due to its persistence and high usage. Three metabolites produced by oxidation of the primary alcohols (forming carboxylates) on the side chains of iopromide were identified in a batch reactor with mixed liquor from a conventional activated sludge. Derivatization of the carboxylic acid to form a methyl ester and interpretation of the MS2 data of this derivative aided in the confirmation of the identities of these metabolites. Furthermore, one metabolite formed by dehydroxylation at the two side chains was identified in a batch reactor with mixed liquor from a nitrifying activated sludge. The MS2 fragmentation pattern of iopromide and its metabolites revealed that the iodinated ring remains intact and that minor transformations in the structure occur during biodegradation of iopromide in biological wastewater treatment plants., This material is based upon work supported by the National Science Foundation under Grant 0233700. S.P. acknowledges a postdoctoral fellowship from the Spanish Ministry of Education, Culture and Science (EX2003-0687).

The key factors governing the absorption of drugs from the lungs are deposition pattern, dissolution rate, solubility and permeability. Whereas the thin epithelium in the respiratory airways is generally considered to constitute a minor barrier in the overall absorption process, the other two components depend, among other parameters, on the formulation and the particle size. This is of particular importance for compounds with very low solubility. The aim of this work was to evaluate strategies to improve the rate and extent of lung absorption in rats which were administered with suspensions of low-solubility compound by the intratracheal route. The two approaches evaluated were (a) the reduction of the particle size by micronization to increase the total surface area and hence to speed up the dissolution, and (b) the addition of methylcellulose to the vehicle, thereby enhancing its viscosity and potentially increasing the compound solubility. For the pharmacokinetic studies performed with male Wistar rats, the test compounds were formulated in PBS (pH 7.4)/0.2 % Tween-80 with or without the addition of 0.3 % methylcellulose (Sigma-Aldrich M0512, 4000 cPs). Compounds were used either directly as obtained from the chemical synthesis or after a micronization process. Suspensions were delivered into the respiratory tract of the rats using a Penn-Century liquid microsprayer (200 uL). Disappearance of the compounds from the target organ within a 24-h period post-dose was monitored by UPLC-ESI-MS/MS. The pharmacokinetic data obtained for chemically diverse compounds indicated that both strategies affected in a very distinct, compound-dependent fashion the pulmonary absorption. In conclusion, micronization and, if this is not technically feasible, addition of methylcellulose to the vehicle may constitute a suitable means of enhancing the pulmonary absorption of compounds delivered into airways of rats. Based on the presented findings, predicting the effect of either methodology appears to be difficult and thus requires a case-by-case evaluation.

Background Testing for cerebrospinal fluid (CSF)-restricted oligoclonal bands (OCB) by isoelectric focusing is used to detect intrathecally produced total IgG. By contrast, antibody indices (AI) are assessed to test for intrathecally produced antigen-specific IgG. A number of previous cases reports have suggested that AI testing might be more sensitive than OCB testing in detecting intrathecal IgG synthesis. Findings Here we report on 21 patients with positive AI for either herpes simplex virus, varicella zoster virus, cytomegalovirus, measles virus, rubella virus, or Borrelia burgdorferi in the absence of total-IgG OCB and, accordingly, in the presence of a normal total-IgG CSF/serum ratio. Conclusion Our findings indicate that AI testing should not generally be omitted in OCB-negative patients and provide a rationale for systematic and prospective studies on the comparative sensitivity and specificity of AI and total-IgG OCB testing in infectious and other diseases of the CNS.

Objectives and Background . Severe primary tricuspid regurgitation in the adult is a rare finding. This study describes the diagnostic findings and the treatment of an isolated congenital cleft of the anterior leaflet of the tricuspid valve as the morphologic substrate for severe tricuspid regurgitation. Methods . The clinical, echocardiographic findings and the follow-up findings of five patients (all male, 20 to 56 years old) with this disorder are described. Four of the five patients underwent cardiac surgery that confirmed the diagnosis. Results . In three of five patients, exertional fatigue was the limiting symptom (New York Heart Association functional classes II and III). The clinical findings included a holosystolic murmur and supraventricular arrhythmias in all patients. Cardiac catheterization, performed in four patients, yielded the incorrect diagnosis of Ebstein's anomaly in three. In one pattent the cleft was associated with an atrial septal defect of the secundum type. In four of five patients successful reconstruction of the tricuspid valve with a DeVega annuloplasty was performed. One patient had a partial excision of the right atrium, and one had a closure of a coexisting atrial septal defect. One patient refused operation. Conclusions . Tricuspid valve anomalies can be accurately identified by Doppler echocardiography. Surgical repair is the treatment of choice in patients with severe tricuspid regurgitation due to a congenital cleft of the anterior leaflet of the tricuspid valve.

The aerobic biodegradability of four antimicrobials (sulfamethazine, sulfamethoxazole, sulfathiazole, and trimethoprim) was investigated in sewage collected at four treatment stages (primary treatment, activated sludge treatment, aerobic nitrification process, and after disinfection of treated sewage) of a municipal sewage treatment plant. The biodegradability tests were conducted in aerated batch reactors by spiking the sewage with 20 microg/L of each of the test substance. Concentration profiles of the assayed compounds were monitored during a 54-d period using liquid chromatography/electrospray ionization/mass spectrometry. Substantial differences in the degradation curves were observed between trimethoprim and the three sulfonamides. The behavior of the latter was characterized by a general biodegradability in the primary and secondary treatment. The highest degradation rates were obtained in the sewage from the activated sludge treatment, where no adaptation phase was observed. On the other hand, the onset of biodegradation in the sewage from the primary treatment was preceded by a lag phase ranging from 10 to 15 d. In contrast, trimethoprim displayed high resistance to microbial degradation in the sewage from the primary treatment and the activated sludge treatment. However, primary degradation of this compound was completed within only 3 d in the sewage from the nitrification process.

In the present study 12 patients with severe chronic heart failure (7 with dilated cardiomyopathy and 5 with ischaemic heart disease) underwent right heart catheterization at rest and during isometric exercise by handgrip before and 6 months after treatment with captopril (mean dose 2 X 39.6 mg/day) while concomitant digitalis and diuretics were unchanged. After 6 months resting haemodynamics changed as follows: decrease in heart rate, mean arterial pressure, pulmonary capillary pressure and total vascular resistance; increase in stroke volume index and stroke work index. During handgrip the following changes were observed after captopril: decrease in total vascular resistance; increase in stroke volume index and stroke work index. Before captopril 11 patients showed no increase or even a decrease in stroke work index during isometric exercise, whereas after captopril 5 out of the 12 patients showed an increase in stroke work index. The results demonstrate that in patients with severe chronic heart failure long-term treatment with captopril lowers preload and afterload, which is accompanied by a significant improvement in left ventricular performance at rest. Furthermore, during isometric exercise after captopril an improvement in left ventricular function was found in 5 out of 12 patients.

We present the case of a 75-year-old female with weight loss, anemia, systemic signs of inflammation, mild renal insufficiency, microscopic hematuria, mixed glomerular and tubular proteinuria, and high titers of PR3-ANCA. Renal biopsy demonstrated interstitial nephritis with some sclerosed but otherwise normal glomeruli. Extensive work-up showed no signs of granulomatous inflammation or other vasculitic organ involvement. We presumed this to be a rare renal manifestation of ANCA-associated disease with the presence of sclerosed glomeruli suggesting a previous history of glomerular involvement. In view of the absence of active vasculitic or granulomatous disease, treatment was limited to low-dose corticosteroids with good response.