Distinct serum and cerebrospinal fluid cytokine and chemokine profiles in autoantibody-associated demyelinating diseases

Background: Demyelinating diseases of the central nervous system associated with autoantibodies against aquaporin-4 and myelin-oligodendrocyte-glycoprotein are mediated by different immunopathological mechanisms compared to multiple sclerosis. Objective: The purpose of this study was to evaluate serum and cerebrospinal fluid cytokine/chemokine profiles in patients with autoantibodies against aquaporin-4 or autoantibodies against myelin-oligodendrocyte-glycoprotein-associated demyelination compared to multiple sclerosis and autoimmune encephalitis. Methods: Serum and cerebrospinal fluid cytokine/chemokine levels were analysed using Procartaplex Multiplex Immunoassays. First, we analysed a panel of 32 cytokines/chemokines in a discovery group (nine aquaporin-4-antibody seropositive, nine myelin oligodendrocyte glycoprotein-antibody seropositive, eight encephalitis, 10 multiple sclerosis). Significantly dysregulated cytokines/chemokines were validated in a second cohort (11 aquaporin-4-antibody seropositive, 18 myelin oligodendrocyte glycoprotein-antibody seropositive, 18 encephalitis, 33 multiple sclerosis). Results: We found 11 significantly altered cytokines/chemokines in cerebrospinal fluid and serum samples in the discovery group (a proliferation-inducing ligand, fractalkine=CX3CL1, growth-regulated oncogene-\u3b1, interleukin-1 receptor antagonist, interleukin-6, interleukin-8=CXCL8, interleukin-10, interleukin-21, interferon-&3-induced protein-10=CXCL10, monokine induced by interferon-&3=CXCL9, macrophage inflammatory protein-1 f=CCL4). Most of these cytokines/chemokines were up-regulated in autoantibodies against aquaporin-4 or autoantibodies against myelin-oligodendrocyte-glycoprotein positive patients compared to multiple sclerosis. We confirmed these results for cerebrospinal fluid interleukin-6 and serum interleukin-8, growth-regulated oncogene-\u3b1, a proliferation-inducing ligand and macrophage inflammatory protein-1\u3b2 in the validation set. Receiver-operating characteristic analysis revealed increased levels of cerebrospinal fluid interleukin-6, serum interleukin-8 and growth-regulated oncogene-\u3b1 in most patients with autoantibody-associated neurological diseases. Conclusion: This study suggests that distinctive cerebrospinal fluid and serum cytokine/chemokine profiles are associated with autoantibody-mediated demyelination, but not with multiple sclerosis

Detection Methods for Autoantibodies in Suspected Autoimmune Encephalitis

This review provides an overview on different antibody test methods that can be applied in cases of suspected paraneoplastic neurological syndromes (PNS) and anti-neuronal autoimmune encephalitis (AIE) in order to explain their diagnostic value, describe potential pitfalls and limitations, and discuss novel approaches aimed at discovering further autoantibodies. Onconeuronal antibodies are well-established biomarkers for PNS and may serve as specific tumor markers. The recommended procedure to detect onconeuronal antibodies is a combination of indirect immunohistochemistry on fixed rodent cerebellum and confirmation of the specificity by line assays. Simplification of this approach by only using line assays with recombinant proteins bears the risk to miss antibody-positive samples. Anti-neuronal surface antibodies are sensitive and specific biomarkers for AIE. Their identification requires the use of test methods that allow the recognition of conformation dependent epitopes. These commonly include cell-based assays and tissue based assays with unfixed rodent brain tissue. Tissue based assays can detect most of the currently known neuronal surface antibodies and thus enable broad screening of biological samples. A complementary testing on live neuronal cell cultures may confirm that the antibody recognizes a surface epitope. In patients with peripheral neuropathy, the screening may be expanded to teased nerve fibers to identify antibodies against the node of Ranvier. This method helps to identify a novel subgroup of peripheral autoimmune neuropathies, resulting in improved immunotherapy of these patients. Tissue based assays are useful to discover additional autoantibody targets that play a role in diverse autoimmune neurological syndromes. Antibody screening assays represent promising avenues of research to improve the diagnostic yield of current assays for antibody-associated autoimmune encephalitis

Evaluation of Proximity Ligation Assay in Neurodegenerative Diseases

In den letzten Jahrzehnten erhöhte sich weltweit die Lebenserwartung der Bevölkerung. Mit fortschreitendem Alter steigt die Inzidenz vieler Erkrankungen inklusive neurodegenerativer Erkrankungen wie Morbus Parkinson (PD) und Morbus Alzheimer (AD). Neurodegenerative Erkrankungen (NDDs) kennzeichnen sich durch einen Verlust von Neuronen und die Akkumulation verschiedener physiologischer, jedoch posttranslational modifizierter Proteine aus. Allgemein werden diese Krankheiten durch die Akkumulation eines einzelnen Proteins beschrieben. Allerdings zeigten mehrere Studien ko-lokalisierte Akkumulationen von mehr als nur einem neuropathologischen Protein in derselben pathologischen Struktur. Eine endgültige Diagnose von NDDs kann erst nach dem Tod gestellt werden, wodurch die Aufklärung der zugrundeliegenden Pathomechanismen verhindert wird. Wir verglichen konfokale Immunfluoreszenz-Laser-Scanning-Mikroskopie (LSM) mit dem neuartigen Proximity Ligation Assays (PLA) in histologischen Schnitten von Hirngewebe von Patienten mit verschiedenen NDDs mit mehr als einem pathologischen Protein in den jeweiligen Proteinablagerungen (gemischte Proteinopathien). In weiterer Folge wurden die Ergebnisse mit Fluoreszenz- (Förster-) Resonanz-Energieübertragung (FRET) validiert. In der LSM-Analyse stellte sich heraus, dass die Lasereinstellungen die Anzahl der Protein-Ko-Lokalisierungen signifikant beeinflussen kann. Die PLA-Auswertung zeigte ein größeres Maß an Protein-Protein-Ko-Lokalisierung in menschlichen Hirngewebeabschnitten verschiedener NDDs als der Goldstandard FRET. Wir stellten fest, dass die Verwendung des PLA eine schnelle und bequeme Anwendung darstellt, aber nicht für die Bewertung der Protein-Protein-Ko-Lokalisierung in Fällen von NDDs verwendbar ist. PLA erlaubt keine ausreichende Aussage über Protein-Protein-Ko-Lokalisierungen, da keine weitere Bestimmung des exakten Abstandes unter 40nm erfolgen kann. Zusätzlich ist die Anwendung des PLA auf Gewebeschnitten im Vergleich zu einzelnen Zellen sehr teuer. Für die Ko-Lokalisierung und Interaktionsanalyse ist der PLA nicht geeignet, um FRET, inklusive spezialisierter mathematischer Makroanalyse, als Goldstandard zu ersetzen.Over recent decades life expectancy of the human population increased all around the world. With progressing age the incidence of many diseases like cancer, cardiovascular diseases, and neurodegenerative diseases such as Parkinson disease (PD) and Alzheimer disease (AD) increases. Neurodegenerative diseases (NDDs) are characterized by loss of neurons and accumulation of various physiologically occurring proteins with posttranslational modifications. While typically diseases are described by the accumulation of the respective protein causing the disease, several studies showed co-localizations with at least one more protein in pathologic regions. A definitive diagnosis of NDDs can only be obtained post mortem impeding elucidation of underlying pathomechanisms. To allow evaluation of potential interaction of proteins associated with various NDDs in the same brain (i.e. mixed proteinopathies), we tested the application of the novel proximity ligation assay (PLA) in comparison with confocal immunofluorescence laser scanning microscopy (LSM). To elucidate mere co-localization, we additionally validated our data with Fluorescence (Förster) resonance energy transfer (FRET). LSM analysis revealed that laser settings significantly influence interpretation of the number of identified protein-protein co-localizations. PLA detects more protein-protein co-localization in human brain tissue sections of various NDDs compared to the gold standard FRET. We concluded that the use of PLA is a fast and convenient application but not suitable for evaluation of protein-protein co-localization and protein-protein interaction in cases of NDD. PLA identifies protein-protein proximities of up to 40nm with no further discrimination of co-localization/interaction below 40nm. In comparison FRET analysis can measure protein-protein proximities with a cut-off of 10nm. Also, the use of PLA on tissue sections is associated with high costs. For co-localization and interaction analysis PLA is not suitable to replace FRET with specialized mathematical macro-analysis as gold standard.Vorgelegt von: Carmen SchwaigerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersWien, FH Campus Wien, Masterarb., 2017(VLID)227945

Positron emission tomography in the assessment of left ventricular function in healthy rats: A comparison of four imaging methods

Objective To measure left ventricular (LV) function parameters in heart of healthy rats by three different positron emission tomography (PET) imaging techniques and by magnetic resonance imaging (MRI). Methods ECG-gated microPET examinations were obtained in seven healthy rats with 2-deoxy-2-[18F]fluoro-d-glucose (FDG) for calculation of LV-function from the blood-pool phase of the dynamic recording (FDGBP), and also from the later myocardial uptake (FDGMyo). On subsequent days, we re-measured LV-function using the novel blood-pool tracer 68Ga-albumin (AlbBP) and again by FDG (FDGMyo2) in one setting. Cine-MRI examination provided the reference standard measurement. Results The mean LV ejection fractions (LVEF) were 56 ± 3 (FDGBP), 55 ± 3 (FDGMyo), 56 ± 3 (FDGMyo2), 57 ± 3 (AlbBP), and 57 ± 2 (MRI). There were good to excellent correlations found between the LVEF-values as compared to MRI reference standard for FDGBP (r = 0.71), FDGMyo (r = 0.86) and AlbBP (r = 0.88). Both of the blood-pool methods significantly overestimated the magnitudes of end-diastolic-volume and end-systolic-volume, whereas FDGMyo matched closely to the MRI reference standard. There was no significant bias for both blood-pool methods and a minor negative bias for FDGMyo regarding the LV ejection fraction (LVEF) when compared to cine-MRI results. There was no significant difference between the means of FDGMyo and FDGMyo2 (P = .50). Conclusions Relative to reference standard MRI measurements of LVEF, there was excellent agreement between PET-based measurements, notably for the novel blood-pool tracer 68Ga-albumin

Use of Complementary and Alternative Medicine (CAM) as Part of the Oncological Treatment: Survey about Patients' Attitude towards CAM in a University-Based Oncology Center in Germany.

INTRODUCTION:To understand if and which patients would be open-minded to Complementary and Alternative Medicine (CAM) use parallel to their oncological treatment. Moreover, we sought to determine which methods are most accepted and which are the primary motivators to use CAM. METHODS:We developed and anonymously conducted a questionnaire for patients in the oncology center (TU Munich). Questions focus on different CAM methods, previous experiences, and willingness to apply or use CAM when offered in a university-based setting. RESULTS:A total of 171 of 376 patients (37.4% women, 62.0% men, 0.6% unknown) participated. This corresponds to a return rate of 45%. Median age was 64 years (17-87 years). Of all participants, 15.2% used CAM during their oncological therapy; 32.7% have used it in the past. The majority (81.9%) was not using CAM during therapy; 55.5% have not used CAM in the past respectively. The analysis revealed a significant correlation between education and CAM use during therapy (r = 0.18; p = 0.02), and CAM use in the past (r = 0.17; p = 0.04). Of all patients using CAM during therapy, favored methods were food supplements (42.3%), vitamins/minerals (42.3%), massage (34.6%). Motivations are especially the reduction of side effect and stress, the positive effect of certain CAM-treatments on the immune system and tumor therapy. Results showed no difference between women and men. Most patients not having had any experience with CAM complain about the deficiency of information by their treating oncologist (31.4%) as well as missing treatment possibilities (54.3%). CONCLUSION:Since many patients believe in study results demonstrating the efficacy of CAM, it stresses our task to develop innovative study protocols to investigate the outcomes of certain CAM on symptom reduction or other endpoints. Thus, prospective trials and innovative evidence-based treatment concepts to include CAM into high-end oncology is what patients demand and what a modern oncology center should offer

User motives.

<p>Motives of oncological patients to use CAM (n = 171).</p

User rate of CAM.

<p>User rate of CAM during and before therapy. The percentage for all are calculated with n = 171; for the different units, the percentage of n = 26 / n = 56 are displayed for during and before therapy respectively.</p

Force-Clamp Spectroscopy of Single-Protein Monomers Reveals the Individual Unfolding and Folding Pathways of I27 and Ubiquitin

Single-protein force experiments have relied on a molecular fingerprint based on tethering multiple single-protein domains in a polyprotein chain. However, correlations between these domains remain an issue in interpreting force spectroscopy data, particularly during protein folding. Here we first show that force-clamp spectroscopy is a sensitive technique that provides a molecular fingerprint based on the unfolding step size of four single-monomer proteins. We then measure the force-dependent unfolding rate kinetics of ubiquitin and I27 monomers and find a good agreement with the data obtained for the respective polyproteins over a wide range of forces, in support of the Markovian hypothesis. Moreover, with a large statistical ensemble at a single force, we show that ubiquitin monomers also exhibit a broad distribution of unfolding times as a signature of disorder in the folded protein landscape. Furthermore, we readily capture the folding trajectories of monomers that exhibit the same stages in folding observed for polyproteins, thus eliminating the possibility of entropic masking by other unfolded modules in the chain or domain-domain interactions. On average, the time to reach the I27 folded length increases with increasing quenching force at a rate similar to that of the polyproteins. Force-clamp spectroscopy at the single-monomer level reproduces the kinetics of unfolding and refolding measured using polyproteins, which proves that there is no mechanical effect of tethering proteins to one another in the case of ubiquitin and I27