Plasma derived extracellular vesicle biomarkers of microglia activation in an experimental stroke model.

Chronic microglia activation post-stroke is associated with worse neurological and cognitive outcomes. However, measurement of microglia activation in vivo is currently limited. Plasma derived extracellular vesicles (EVs) are cell-specific indicators that may allow for non-invasive measurement of microglia phenotype. The aim of this study was to identify activation-state specific microglia EVs (MEVs) in vitro followed by validation in an experimental stroke model. Following pro-inflammatory activation, MEVs contain the microglia protein TMEM119 alongside increased expression of the Toll-like receptor 4 co-receptor CD14. Immunoprecipitation followed by fluorescent nanoparticle tracking analysis (ONI Nanoimager) was used to confirm the isolation of TMEM119+/CD14+ EVs from rat plasma. Electron microscopy confirmed that TMEM119 and CD14 localize to the MEV membrane. To model ischemia, plasma was collected from 3-month wildtype Fischer344 rats prior to, 7 and 28 days after endothelin-1 or saline injection into the dorsal right striatum. Fluorescently labelled MEVs were directly measured in the plasma using nanoflow cytometry (Apogee A60 Microplus). We report a significant increase in circulating TMEM119+/CD14+ EVs 28-days post-stroke in comparison to baseline levels and saline-injected rats, which correlated weakly with stroke volume. TMEM119+/MHC-II+ EVs were also increased post-stroke in comparison to baseline and saline-injected animals. This study is the first to describe an EV biomarker of activated microglia detected directly in plasma following stroke and represents a future tool for the measurement of microglia activity in vivo.

Fibrinogen primes the microglial NLRP3 inflammasome and propagates pro-inflammatory signaling via extracellular vesicles: Implications for blood-brain barrier dysfunction.

The brain's response to acute injury is characterized by increased permeability of the blood-brain barrier (BBB) and pro-inflammatory microglia signaling, both of which have been linked to poor cognitive outcomes and neurological disease. The damaged BBB has increased leakiness, allowing serum proteins like fibrinogen into the brain, which interacts with local cells in a deleterious manner. At the same time, in response to injury, microglia demonstrate increased NLRP3 inflammasome activity and heightened release of pro-inflammatory cytokines. The relationship between increased fibrinogen uptake and microglial inflammasome signaling in the injured brain has not been well described. In this work, we investigate fibrinogen mediated NLRP3 inflammasome priming of BV-2 cells and primary adult microglia and propose a role for extracellular vesicles (EVs) as propagators of this interaction. Following exposure to fibrinogen microglia significantly upregulate transcription of IL-1ß, IL-6, NLRP3 and other pro-inflammatory cytokines which was sustained by repeated fibrinogen exposure. Inhibition of fibrinogen mediated NLRP3 signaling was achieved at the transcriptional and assembly level using cannabidiol (CBD) and the NLRP3 inhibitor MCC950, respectively. EVs released following NLRP3 priming carry IL-1ß, IL-18 mRNA and fibrinogen, propagate inflammatory signaling and can be detected in the circulation following BBB disruption in a preclinical stroke model. In conclusion, the interplay between fibrinogen extravasation, microglial NLRP3 signaling, and EV release can perpetuate chronic pro-inflammatory signaling and represents a novel method of inflammatory propagation.

Improving access to affordable quality-assured inhaled medicines in low- and middle-income countries.

BACKGROUND: Access to affordable inhaled medicines for chronic respiratory diseases (CRDs) is severely limited in low- and middle-income countries (LMICs), causing avoidable morbidity and mortality. The International Union Against Tuberculosis and Lung Disease convened a stakeholder meeting on this topic in February 2022.METHODS: Focused group discussions were informed by literature and presentations summarising experiences of obtaining inhaled medicines in LMICs. The virtual meeting was moderated using a topic guide around barriers and solutions to improve access. The thematic framework approach was used for analysis.RESULTS: A total of 58 key stakeholders, including patients, healthcare practitioners, members of national and international organisations, industry and WHO representatives attended the meeting. There were 20 pre-meeting material submissions. The main barriers identified were 1) low awareness of CRDs; 2) limited data on CRD burden and treatments in LMICs; 3) ineffective procurement and distribution networks; and 4) poor communication of the needs of people with CRDs. Solutions discussed were 1) generation of data to inform policy and practice; 2) capacity building; 3) improved procurement mechanisms; 4) strengthened advocacy practices; and 5) a World Health Assembly Resolution.CONCLUSION: There are opportunities to achieve improved access to affordable, quality-assured inhaled medicines in LMICs through coordinated, multi-stakeholder, collaborative efforts.

Ambroxol as a novel disease-modifying treatment for Parkinson's disease dementia: protocol for a single-centre, randomized, double-blind, placebo-controlled trial.

BACKGROUND: Currently there are no disease-modifying treatments for Parkinson's disease dementia (PDD), a condition linked to aggregation of the protein α-synuclein in subcortical and cortical brain areas. One of the leading genetic risk factors for Parkinson's disease is being a carrier in the gene for ß-Glucocerebrosidase (GCase; gene name GBA1). Studies in cell culture and animal models have shown that raising the levels of GCase can decrease levels of α-synuclein. Ambroxol is a pharmacological chaperone for GCase and is able to raise the levels of GCase and could therefore be a disease-modifying treatment for PDD. The aims of this trial are to determine if Ambroxol is safe and well-tolerated by individuals with PDD and if Ambroxol affects cognitive, biochemical, and neuroimaging measures. METHODS: This is a phase II, single-centre, double-blind, randomized placebo-controlled trial involving 75 individuals with mild to moderate PDD. Participants will be randomized into Ambroxol high-dose (1050 mg/day), low-dose (525 mg/day), or placebo treatment arms. Assessments will be undertaken at baseline, 6-months, and 12-months follow up times. Primary outcome measures will be the Alzheimer's disease Assessment Scale-cognitive subscale (ADAS-Cog) and the ADCS Clinician's Global Impression of Change (CGIC). Secondary measures will include the Parkinson's disease Cognitive Rating Scale, Clinical Dementia Rating, Trail Making Test, Stroop Test, Unified Parkinson's disease Rating Scale, Purdue Pegboard, Timed Up and Go, and gait kinematics. Markers of neurodegeneration will include MRI and CSF measures. Pharmacokinetics and pharmacodynamics of Ambroxol will be examined through plasma levels during dose titration phase and evaluation of GCase activity in lymphocytes. DISCUSSION: If found effective and safe, Ambroxol will be one of the first disease-modifying treatments for PDD. TRIAL REGISTRATION: ClinicalTrials.gov NCT02914366, 26 Sep 2016/retrospectively registered.

XAS/DRIFTS/MS spectroscopy for time-resolved operando investigations at high temperature.

The combination of complementary techniques in the characterization of catalysts under working conditions is a very powerful tool for an accurate and in-depth comprehension of the system investigated. In particular, X-ray absorption spectroscopy (XAS) coupled with diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and mass spectroscopy (MS) is a powerful combination since XAS characterizes the main elements of the catalytic system (selecting the absorption edge) and DRIFTS monitors surface adsorbates while MS enables product identification and quantification. In the present manuscript, a new reactor cell and an experimental setup optimized to perform time-resolved experiments on heterogeneous catalysts under working conditions are reported. A key feature of this setup is the possibility to work at high temperature and pressure, with a small cell dead volume. To demonstrate these capabilities, performance tests with and without X-rays are performed. The effective temperature at the sample surface, the speed to purge the gas volume inside the cell and catalytic activity have been evaluated to demonstrate the reliability and usefulness of the cell. The setup capability of combining XAS, DRIFTS and MS spectroscopies is demonstrated in a time-resolved experiment, following the reduction of NO by Rh nanoparticles supported on alumina.

Structural analysis and optical properties of the Bi2-x Y x WO6 system.

Photocatalytic conversion of solar energy into chemical energy has attracted considerable interest for several decades. One compound already reported as a visible-light-active photocatalyst for water splitting is BiYWO6, a member of the Bi2-x Y x WO6 family of compounds. The structural and optical properties of other members of this family have not been reported to date. In this work, we synthesized various compositions of Bi2-x Y x WO6, studied their optical properties, and report their structural parameters obtained by utilizing powder diffraction coupled with Rietveld refinement.

The Time-resolved and Extreme-conditions XAS (TEXAS) facility at the European Synchrotron Radiation Facility: the energy-dispersive X-ray absorption spectroscopy beamline ID24.

The European Synchrotron Radiation Facility has recently made available to the user community a facility totally dedicated to Time-resolved and Extreme-conditions X-ray Absorption Spectroscopy--TEXAS. Based on an upgrade of the former energy-dispersive XAS beamline ID24, it provides a unique experimental tool combining unprecedented brilliance (up to 10(14) photons s(-1) on a 4 µm × 4 µm FWHM spot) and detection speed for a full EXAFS spectrum (100 ps per spectrum). The science mission includes studies of processes down to the nanosecond timescale, and investigations of matter at extreme pressure (500 GPa), temperature (10000 K) and magnetic field (30 T). The core activities of the beamline are centered on new experiments dedicated to the investigation of extreme states of matter that can be maintained only for very short periods of time. Here the infrastructure, optical scheme, detection systems and sample environments used to enable the mission-critical performance are described, and examples of first results on the investigation of the electronic and local structure in melts at pressure and temperature conditions relevant to the Earth's interior and in laser-shocked matter are given.

The time-resolved and extreme conditions XAS (TEXAS) facility at the European Synchrotron Radiation Facility: the general-purpose EXAFS bending-magnet beamline BM23.

BM23 is the general-purpose EXAFS bending-magnet beamline at the ESRF, replacing the former BM29 beamline in the framework of the ESRF upgrade. Its mission is to serve the whole XAS user community by providing access to a basic service in addition to the many specialized instruments available at the ESRF. BM23 offers high signal-to-noise ratio EXAFS in a large energy range (5-75 keV), continuous energy scanning for quick-EXAFS on the second timescale and a micro-XAS station delivering a spot size of 4 µm × 4 µm FWHM. It is a user-friendly facility featuring a high degree of automation, online EXAFS data reduction and a flexible sample environment.

Serum homocysteine, folate, vitamin B12 levels and arterial stiffness in diabetic patients: which of them is really important in atherogenesis?

AIM: Hyperhomocystinaemia is associated with macro- and microangiopathic diabetic complications. However, the role of homocysteine (Hcy), serum folate, and vitamin B12 level in the development of premature vascular damage in type 2 diabetic patients is not clear. The present study was designed to assess the relationship between total Hcy, folate, and vitamin B12 levels and arterial stiffness, an early marker of generalized atherosclerosis. METHODS: As many as 86 subjects with type 2 diabetes mellitus were studied. All participants were evaluated for glucose, HbA(1C), lipid profile, hs-CRP, endothelin, Hcy, vitamin B12, and folate. Pulse wave velocity (PWV) and augmentation index (AI) were performed as a non-invasive recording and computer analysis of the two artery sites pressure waveform using SphygmoCor (version 7.1, AtCor Medical, Sydney, Australia). RESULTS: Hcy was significantly positively associated with age, serum creatinine, and vitamin B12 levels. No association between Hcy and folate was observed. The Hcy concentration was significantly positively associated with PWV (r = 0.540, p < 0.0001) and AI (r = 0.390, p < 0.0001). In a general linear model of PWV, Hcy emerged as an independent predictor of PWV even after controlling for age, creatinine, vitamin B12, and folate levels. In a multiple linear regression analysis, the association between Hcy and arterial stiffness was independent of traditional cardiovascular risk factors. Vitamin B12 levels were significantly inversely associated with tHcy (r = - 0.263, p = 0.015) and marginally associated with PWV(r = - 0.212, p = 0.052). Significant associations between folate levels and PWV were not detected. CONCLUSIONS: The results lend support to the hypothesis that elevated Hcy may have a key role in the development of atherogenesis in diabetic patients. Additionally, vitamin B12 is significantly associated with tHcy concentrations and is identified as a marginally independent correlate of PWV in diabetic patients in the absence of folate deficiency.

XMCD under pressure at the Fe K edge on the energy-dispersive beamline of the ESRF.

The present paper demonstrates the feasibility of X-ray absorption spectroscopy (XAS) and X-ray magnetic circular dichroism (XMCD) at high pressure at the Fe K edge on the ID24 energy-dispersive beamline of the ESRF. In 3d transition metals, performing experiments at the hard X-ray K edge rather than at the magnetically interesting soft X-ray L edges represents the only way to access the high-pressure regime obtainable with diamond anvil cells. The simultaneous availability of a local structure (XAS) and of a magnetic (XMCD) probe on the sample under identical thermodynamical conditions is essential for studying correlations between local structural and magnetic properties. The state-of-the-art theoretical understanding of K-edge XMCD data is briefly summarized, and the set-up of beamline ID24 for high-pressure XMCD experiments is illustrated and the conditions required to perform measurements at the K edges of 3d transition metals are underlined. Finally, two examples of recent high-pressure results at the Fe K edge in pure Fe and Fe3O4 powder are presented.

Exfoliation syndrome: clinical and genetic features.

We have ascertained a large number of individuals and families with exfoliation syndrome in order to clarify the disorder's mode of inheritance. Patients with exfoliation syndrome and their relatives were recruited from the practices of a group of ophthalmologists in Maritime Canada. The degree to which the subjects were affected was graded according to a standardized 1-4-point clinical scheme. Pedigrees were constructed from information supplied by family members and from genealogical sources. A total of 782 patients and relatives participated, of whom 467 were definitely affected. The mean age of affected males and females did not differ significantly, but females appeared to be more severely affected at ascertainment than males. More than half of the affected subjects had definite exfoliation in only one eye. Approximately 30 multiplex families were discovered, including one containing 23 affected members among a total of 137 examined individuals that constitutes the largest exfoliative pedigree thus far described. We observed well-documented paternal transmission of the trait, a finding that has not to our knowledge been previously reported. Clustering of cases in the families provides evidence for the involvement of genetic factors. The possibility of homozygosity is suggested in a few patients by the earlier or more frequent presentation of the disorder in the offspring of two affected parents or consanguineous pairings. Although a multifactorial mode of inheritance cannot be excluded, exfoliation syndrome appears to be inherited as an autosomal dominant trait whose late onset and incomplete penetrance poses a significant but not insuperable obstacle to pedigree construction.

Doctor-patient concordance and patient initiative during episodes of low back pain.

Doctor-patient concordance and patient initiative were examined in a prospective network interview study, with telephone follow-up, of a cohort of 100 patients presenting with low back pain to their family physician. The average overall rate of concordance was 60% (95% CI = 53 to 66), with the highest rates for radiographic imaging studies and sick leave. No correlation was found between concordance and patient parameters. Subjects initiated an average of two (95% CI = 1.7 to 2.3) diagnostic or therapeutic procedures, the most common of which were for medications (40%), followed by bed rest (26%) and back school (22%). One out of every six subjects initiated a referral to a complementary therapist. Positive correlation was found between patient initiatives and pain severity (P = 0.022) and disability (P = 0.02). There was a negative correlation between the subjects' initiatives and their belief that the physician understood the cause of their pain and its influence on their life (P = 0.02). Overall, those patients who described more pain or disability sought more types of diagnostic and therapeutic measures, while those who felt they had been understood sought less.

Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel.

The novel locus Prnd is 16 kb downstream of the mouse prion protein (PrP) gene Prnp and encodes a 179 residue PrP-like protein designated doppel (Dpl). Prnd generates major transcripts of 1.7 and 2.7 kb as well as some unusual chimeric transcripts generated by intergenic splicing with Prnp. Like PrP, Dpl mRNA is expressed during embryogenesis but, in contrast to PrP, it is expressed minimally in the CNS. Unexpectedly, Dpl is upregulated in the CNS of two PrP-deficient (Prnp(0/0)) lines of mice, both of which develop late-onset ataxia, suggesting that Dpl may provoke neurodegeneration. Dpl is the first PrP-like protein to be described in mammals, and since Dpl seems to cause neurodegeneration similar to PrP, the linked expression of the Prnp and Prnd genes may play a previously unrecognized role in the pathogenesis of prion diseases or other illnesses.

A tandem solid phase extraction, reversed-phase HPLC method for determining SDZ WAG 994 in dog, monkey and rat blood.

The development and validation of a sensitive and specific HPLC method for SDZ WAG 994 (I) in dog, monkey and rat blood is described. Sample preparation entailed double solid phase extraction (SPE) of I and the internal standard from 0.5 ml of animal blood using a phenyl and propyl sulfonic acid cation exchange column, sequentially. Chromatographic separation was achieved on a YMC Basic C-8 narrowbore HPLC column and the eluates were detected by UV absorption at 266 nm. The method has a linear response up to at least 1800 ng/ml with a limit of quantification of 1 ng/ml across all species. Analysis of 'blinded' quality control dog and monkey blood samples over 3 or 4 days produced median precisions of 2.89 and 4.77%, and median reproducibilities of 4.86 and 10.9%, respectively. Curve fitting of variability estimates indicated that concentration independent error contributed 3-9% of the total method error for the tandem SPE procedure. Extracted endogenous material from blood matrices, several potential metabolites and cyclohexyladenosine were well resolved from the peaks of interest. The stability of I in dog blood stored at -20 degrees C is at least 6 months. The overall absolute and relative recovery of I using the tandem SPE procedure was 85.5 +/- 5.1% and 96.5 +/- 5.0%, respectively. The ruggedness of the method has been demonstrated by multiple analyses, from several toxicokinetic studies, performed by different analysts using comparable instrumentation.

Monoclonal origin of localised orbital amyloidosis detected by molecular analysis.

AIMS: Primary localised orbital amyloidosis is a rare disease. The purpose of this study was to describe two cases of primary orbital amyloidosis and emphasise the value of molecular analysis of immunoglobulin gene rearrangement in identifying a monoclonal population of cells responsible for the amyloid production. METHODS: Charts and biopsy specimens of each case were reviewed. Conventional light microscopy, immunohistochemistry, and polymerase chain reaction (PCR) analysis for immunoglobulin gene rearrangement were performed in both cases. RESULTS: An unusual presentation of localised primary amyloidosis with bilateral and extensive enlargement of multiple extraocular muscles was seen in case 1. The presence of amyloid deposits was confirmed by biopsy in both cases. Evidence of a monoclonal population of plasma cells was shown by immunohistochemical analysis in case 2 only. The monoclonal origin of the cells responsible for the amyloid deposition was determined by PCR analysis demonstrating immunoglobulin heavy chain gene rearrangement in both cases. CONCLUSIONS: A monoclonal population of plasma cells responsible for the amyloid deposition was present in these two cases. PCR analysis is extremely helpful in determining monoclonality, a finding that may have important therapeutic and prognostic implications.

Enchondroma of the orbit.

PURPOSE: To report a documented case of orbital enchondroma. METHODS: A 25-year-old woman had an asymptomatic orbital mass. Computed tomographic scan was performed. The mass was surgically removed from the medial wall of the orbit, and the specimen was submitted for histopathologic examination. RESULTS: The tumor showed typical radiologic and histopathologic appearance of an enchondroma with typical hypocellular lobules of cartilage enclosed by lamellar bone. CONCLUSION: Cartilaginous neoplasms in the orbit are extremely rare.

Developmental toxicity of EDS recycle solvent and fuel oil.

Direct coal liquefaction is one of several technologies currently under development as alternative means to produce liquid fuels. Relatively high levels of polycyclic aromatic hydrocarbons are present in distillate fractions boiling above approximately 370 degrees C. Coal-derived liquids containing substantial amounts of material from this boiling range were genotoxic in in vitro tests and carcinogenic in mouse skin. Some of the liquids were also teratogenic in rodents. The present report describes studies which assessed the potential effects of 2 coal-derived liquids, recycle solvent (nominal boiling range 200-427 degrees C) and an experimental industrial fuel oil (nominal boiling range 204-538 degrees C) on prenatal development in the rat. The test materials were produced by the EDS direct coal liquefaction process and contained substantial amounts of material boiling above 370 degrees C. Test materials were administered by gavage to pregnant female Sprague-Dawley rats from days 6 to 19 of gestation (G). Animals were sacrificed on day 20G and the uterine contents were removed and examined. Results of both studies were similar. The number of live fetuses declined in a dose-related manner, and there was evidence of intrauterine growth retardation in fetuses which survived to day 20G. Statistically significant effects were noted at doses which did not appear to be maternally toxic. The frequency of malformation was not significantly elevated in either study; however, a thorough evaluation of this endpoint was precluded by embryo lethality at the high doses. It was apparent that both of the EDS liquids examined affected prenatal survival and growth. However, in contrast to studies of other coal-derived liquids, there was no evidence of teratogenic effects at non-toxic doses.

The Memorial Pain Assessment Card. A valid instrument for the evaluation of cancer pain.

Effective evaluation and treatment of cancer pain require valid and independent measurement of pain intensity, pain relief, and psychological distress. The Memorial Pain Assessment Card (MPAC) is a simple instrument designed to provide rapid evaluation of these subjective experiences. On the 8.5 by 11 inch card are printed the eight pain intensity descriptors, and three visual analog scales which measure pain intensity, pain relief, and mood. Experienced patients can complete it in less than 20 seconds. The authors administered the MPAC to 50 hospitalized cancer patients within 48 hours of referral to the Pain Service for inadequate pain control, together with standard measures: The McGill Pain Questionnaire, Profile of Mood States, Hamilton Depression Scale, and Zung Anxiety Scale. Correlational and multiple regression analyses revealed that the MPAC can distinguish pain intensity from pain relief and from general psychological distress, and it can provide multidimensional assessment that is practically equivalent to the full assessment battery. We conclude that the MPAC is valid and effective for clinical use, and recommend it for the assessment of individual patients, and as an outcome measure in clinical trials.