This study investigates the suitability of beta-2-microglobulin (β2-microglobulin), lactate and angiotensin-converting enzyme (ACE) as biomarkers, given the good availability of these parameters in routine diagnostics but lack of data in this regard. For this purpose, 6,310 CSF samples obtained at the Neurological Clinic of the University Hospital of Würzburg were analyzed. Closer analysis was carried out of 276 cases with non-inflammatory neurological diseases (NIND; control group) and 438 MS cases not taking an immunotherapy treatment (study group). In the MS cases, the form of progression of the disease and the disease activity (clinical relapses, progression index) were recorded. A clear correlation could be seen between age and CSF levels of β2-microglobulin, lactate and ACE in both the MS and control groups, whereby a correction was required for the subsequent comparison studies; this could also at least partly explain the contradictory data obtained in other studies to date. The MS cases showed elevated β2-microglobulin and lactate levels and decreased ACE levels in CSF compared to the controls. In both groups, there was a positive correlation between β2-microglobulin and ACE levels. In the separate analysis of the forms of progression of MS, cases with clinically-isolated syndrome (CIS) and relapsing-remitting MS (RRMS) revealed elevated β2-microglobulin levels, whilst cases with secondary-progressive or primary-progressive MS (SPMS or PPMS) did not. Lactate levels were only increased in cases of CIS. Cases with a relapsing course showed reduced ACE levels. The disease activity could not reliably be mapped by the parameters. Lactate levels tended to be elevated during a relapse, but this result was no longer significant after correction. Lactate levels also showed a positive correlation with the progression index. Our findings in this study provide evidence that the examined analysis parameters cannot be used in isolation to assess progression, disease activity and duration of disease. However, the significant differences between relapsing and chronic-progressive courses support the hypothesis of different underlying mechanisms of pathogenesis, and could serve as a starting basis for further studies. / In der vorliegenden Arbeit wurde die Eignung der im Rahmen der Routinediagnostik verfügbaren, aber unzu¬reichend charakterisierten Analyten Beta-2-Mikroglobulin (β2-Mikroglobulin), Laktat und Angiotensin-Converting Enzyme (ACE) als Biomarker untersucht. Dazu wurden 6.310 an der Neurologischen Klinik des Universitätsklinikums Würzburg gewonnene Liquorproben analysiert. Näher analysiert wurden 276 Fälle mit nicht entzünd¬lichen neurologischen Erkrankungen (NIND; Kontrollgruppe) und 438 nicht immuntherapeutisch behandelte MS-Fälle (Untersuchugsgruppe). Bei den MS-Fällen wurde die Verlaufs¬form und Krankheitsaktivität (klinische Schübe, Progressionsindex) dokumentiert. Es zeigte sich eine deutliche Altersabhängigkeit der Liquorspiegel von β2-Mikroglobulin, Laktat und ACE in der MS- und Kontrollgruppe, was für die sich anschließenden weiteren Vergleichsuntersuchungen eine Korrektur erforderte und zumindest teilweise die wider¬sprüchliche Datenlage bisheriger Studien erklären könnte. MS-Fälle zeigten im Liquor im Vergleich zu Kontrollen erhöhte β2-Mikroglobulin- und Laktat- sowie er¬niedrigte ACE-Spiegel. In beiden Gruppen korrelierten die β2-Mikroglobulin- und ACE-Spiegel positiv miteinander. Bei der getrennten Analyse der MS-Verlaufsformen zeigten Fälle mit klinisch isoliertem Syndrom (CIS) und schubförmig remittierender MS (RRMS) erhöhte β2-Mikroglobulin-Spiegel, Fälle mit sekundär bzw. primär pro¬gredienter MS (SPMS bzw. PPMS) dagegen nicht. Die Laktat-Spiegel waren lediglich bei CIS-Fällen erhöht. Fälle mit schubförmigen Verläufen zeigten reduzierte ACE-Spiegel. Die Krankheitsaktivität wurde durch die Parameter nicht zuverlässig abgebildet. Die Laktat-Spiegel waren tendenziell bei einem Schub erhöht, das Ergebnis war nach Korrektur aber nicht mehr signifikant. Die Laktat-Spiegel korrelierten zudem positiv mit dem Progressionsindex. Die vorliegenden Befunde belegen, dass die untersuchten Analyten alleine nicht in der Lage sind, die Verlaufsform, Krankheitsaktivität und -dauer zu beurteilen. Die deutlichen Unterschiede zwischen schubförmigen und chronisch progredienten Verläufen unterstützen jedoch die Hypothese unterschiedlicher zugrundeliegender Pathomechanismen und könnten als Ausgangspunkt für weitere Untersuchungen dienen.

No description available.

No / Previously, dose emission below 30 L min(-1) through DPI has not been routinely determined. However, during routine use some patients do not achieve 30 L min(-1) inhalation flows. Hence, the aim of the present study was to determine dose emission characteristics for low inhalation flows from terbutaline sulphate Turbuhaler. Total emitted dose (TED), fine particle dose (FPD) and mass median aerodynamic diameter (MMAD) of terbutaline sulphate Turbuhaler were determined using inhalation flows of 10-60 L min(-1) and inhaled volume of 4 L. TED and FPD increase significantly with the increase of inhalation flows (p <0.05). Flows had more pronounced effect on FPD than TED, thus, faster inhalation increases respirable amount more than it increases emitted dose. MMAD increases with decrease of inhalation flow until flow of 20L min(-1) then it decreases. In vitro flow dependent dose emission has been demonstrated previously for Turbuhaler for flow rates above 30 L min(-1) but is more pronounced below this flow. Minimal FPD below 30 L min(-1) suggests that during routine use at this flow rate most of emitted dose will impact in mouth. Flow dependent dose emission results suggest that Pharmacopoeias should consider the use variety of inhalation flows rather than one that is equivalent to pressure drop of 4 KPa.

Indivíduos portadores do glutamato na posição 27 do gene que codifica para o receptor beta2-adrenérgico têm resposta vasodilatadora muscular aumentada durante manobras fisiológicas. No entanto, o impacto do consumo agudo de gordura nessa resposta não é conhecido. Neste estudo, testou-se a hipótese de que o consumo gordura afetaria a resposta vasodilatadora aumentada destes indivíduos durante manobras fisiológicas. Vinte e cinco indivíduos saudáveis foram subdivididos em dois grupos: 11 homozigotos para o glutamato (Glu27Glu, 40+-3 anos; 65+-3kg) e 14 homozigotos para a glutamina (Gln27Gln, 40+-2 anos; 64+-2kg). O fluxo sangüíneo muscular foi medido por pletismografia de oclusão venosa. A resposta vasodilatadora muscular foi avaliada durante 3 minutos de exercício e estresse mental em jejum e 3 horas após consumo de 62 g de gordura. A condutância basal foi semelhante entre grupos (Glu27Glu=2,3+-0,1; Gln27Gln=2,2+-0,1; P=0,21). O aumento da condutância vascular durante exercício e durante o estresse mental foi maior no grupo Glu27Glu (0,73+-0,2 vs 0,22+-0,1; P=0,008 e 1,8?0,3 vs 1,2+-0,2; P=0,04, respectivamente). O consumo agudo de uma preparação rica em gordura eliminou esta diferença. A resposta de pressão arterial e freqüência cardíaca foi semelhante antes e após a ingestão de gordura. Os níveis de triglicérides, glicose e insulina foram semelhantes ao longo de todo período de estudo. O consumo agudo de gordura elimina a resposta aumentada do fluxo sangüíneo muscular durante manobras fisiológicas dos indivíduos portadores do genótipo Glu27Glu no receptorbeta2- adrenérgico. / Subjects who have glutamic acid at position 27 in gene encoding to beta2-adrenoceptor have increased muscle vasodilatory response during physiological maneuvers. However, the impact of a high-fat meal in this response is unknown. We tested the hypothesis that a high-fat meal would modify the increased muscle vascular reactivity during handgrip and mental stress in these subjects. Twenty-five healthy subjects were subdivided in two groups: 11 were homozygous to glutamic acid (Glu27Glu, 40?3 years; 65+-3kg) and 14 were homozygous to glutamine (Gln27Gln, 40+-2 years; 64+-2kg). Forearm blood flow was measured by venous occlusion pletysmography. Forearm blood flow was recorded for 3 minutes of handgrip and mental stress during fasting and three hours after 62g of fat consumption. Baseline forearm vascular conductance was similar between groups (Glu27Glu=2.3+-0.1; Gln27Gln=2.2+-0.1; P=0.21). Forearm vascular conductance during handgrip and mental stress was greater in the genotype Glu27Glu (0.73+-0.2 vs 0.22+-0.1; P=0.008 and 1.8+-0.3 vs 1.2+-0.2; P=0.04, respectively). Acute fat consumption eliminated the difference of vasodilatory response previously achieved. Blood pressure and heart rate response were similar before and after fat intake. Triglycerides, glucose and insulin levels were also similar between groups. We concluded that high-fat ingestion abolishes the augmented muscle blood flow responses during physiological maneuvers in individuals who are homozygous for the Glu27 allele of the beta2-adrenoceptor gene.

Die Funktionalität β1- und β2-adrenerger Rezeptoren wird durch Polymorphismen in ihrer kodierenden Region moduliert. Wir haben uns die Technik des Fluoreszenz-Resonanz- Energie-Transfers (FRET) zu Nutze gemacht, um den Einfluss der am häufigsten vorkommenden Polymorphismen (Ser49Gly und Gly389Arg im β1AR, Arg16Gly und Gln27Glu im β2AR) auf die Rezeptorkonformation nach Aktivierung zu untersuchen. Dafür wurden FRET-Sensoren für die beiden βAR-Subtypen mit einem gelb-fluoreszierenden Protein (YFP) sowie einem cyan-fluoreszierenden Protein (CFP oder Cerulean) in der dritten intrazellulären Schleife bzw. am C-Terminus verwendet. Nach Stimulierung der βARSensoren konnte die Aktivierung der polymorphen Rezeptorvarianten in lebenden Zellen in Echtzeit untersucht werden. Dabei behielten die FRET-Sensoren sowohl die Bindungsaffinitäten der nativen Rezeptoren als auch eine intakte Funktionalität hinsichtlich der Bildung von sekundären Botenstoffen. Der Vergleich der Aktivierungskinetiken der verschieden polymorphen Varianten des β1AR und β2AR ergab keine signifikanten Unterschiede nach einer einmaligen Stimulation. Es zeigte sich jedoch, dass Rezeptorpolymorphismen die Aktivierungskinetik vorstimulierter βAR erheblich beeinflussen. So konnten wir im Vergleich zur ersten Aktivierung eine schnellere Aktivierung der Gly16-Varianten des β2AR sowie des Gly49Arg389-β1AR feststellen, während die Arg16-β2AR-Variante und der Ser49Gly389-β1AR dagegen bei einer wiederholten Stimulation langsamer aktiviert wurden. Diese Ergebnisse lassen auf ein "Rezeptorgedächtnis" schließen, das spezifisch für bestimmte polymorphe Rezeptorvarianten ist und eine βAR-Subtyp-spezische Ausprägung zeigt. Die Ausbildung der unterschiedlichen Aktivierungskinetiken hing von der Interaktion des Rezeptors mit löslichen intrazellulären Faktoren ab und bedurfte einer Phosphorylierung intrazellulärer Serin- und Threonin-Reste durch G-Protein-gekoppelte Rezeptorkinasen. Die Interaktion mit löslichen intrazellulären Faktoren scheint für den β1AR weniger stark ausgeprägt zu sein als für den β2AR. Die cAMP-Produktion war für die schneller werdenden, “hyperfunktionellen” Gly16-β2ARVarianten signifikant um mehr als 50% höher im Vergleich zur “hypofunktionellen” Arg16- Variante. Die unterschiedliche Funktionalität spiegelte sich im Therapieausgang bei Tokoysepatientinnen wider, dessen Erfolg mit dem Arg16Gly Polymorphismus verknüpft war. Die Daten implizieren eine intrinsische, polymorphismusabhängige Eigenschaft der βAR, die die Aktivierungskinetik der Rezeptoren bei wiederholten Stimulationen determiniert. Diese könnte auch für die zwischen Individuen variierende Ansprechbarkeit auf β-Agonisten und β-Blocker mitverantwortlich sein. / Signaling through G protein-coupled receptors is known to be influenced by receptor polymorphisms, yet the molecular basis for the functional differences is unclear. To investigate the impact of the most frequent polymorphic sites of the β1- and the β2– adrenergic receptor (Ser49Gly and Gly389Arg for β1AR, Arg16Gly and Gln27Glu for β2AR) on receptor conformation we used a fluorescence resonance energy transfer (FRET) based approach. We made use of βAR-FRET sensors with a yellow fluorescent protein (YFP) inserted into the third intracellular loop and a cyan fluorescent protein (CFP or Cerulean) fused to the C-terminal tail of the βAR. These sensors retained key pharmacological and functional characteristics of the native receptors. Upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells and found that βAR respond to repeated activation with a change of their activation kinetics during subsequent stimulations. This phenomenon differed between polymorphic variants of the βAR. The “hyperfunctional” Gly16-β2AR variants as well as the Gly49Arg389-β1AR became faster in their activation kinetics, while the “hypofunctional” Arg16-β2AR and the Ser49Gly389-β1AR became slower compared to their initial activation. These differences depended on the interaction with soluble cytosolic factors that occurred after the initial activation, and on the phosphorylation of agonist-bound receptors through G protein-coupled receptor kinases. The “memory“ of previous activation is formed already after a first stimulation of only five seconds, whereas the β1AR memory necessitates prestimulation for five minutes and seems to be based on a less stable interaction with intracellular proteins compared to the β2AR. Assuming short-lived and repetitive receptor-ligand interaction under native conditions, we hypothesized that faster activation during single ligand-receptor interaction represents the basis for more effective signaling to downstream effectors. Indeed, the extent of cAMP formation was enhanced by 50% upon stimulation of the Gly16-β2AR compared to the Arg16 variant. The different functionality reflected the outcome of tocolysis treatment with the β2-agonist fenoterol whose success correlated with the Arg16Gly genotype of the patients. Our findings suggest an intrinsic, polymorphism-specific property of the βAR that alters activation kinetics upon continued stimulation and that might account for individual drug responses.

La Beta-2-microglobuline est une protéine de 12kDa, impliquée dans une maladie dûe à un mauvais repliement: l'amylose liée à la dialyse. Elle constitue donc un modèle pour la formation de fibrilles amyloides et pour le repliement des protéines. La B2M est un objet à la fois difficile et fructueux à étudier. La production de B2M est complexe et demande une optimisation important pour obtenir une protéine correctement repliée et atteindre des rendements approprié pour des études de RMN et SAXS. Le repliement de la B2M est sensible au solvent, à la température, à la concentration et souvent aux conditions de préparation. Pourtant notre étude, à l'aide de plusieurs méthodes biophysiques, a pu révéler plusieurs faits essentiels de son mécanisme de repliement et de la structure et propriétés des intermédiaires. Un premier résultat est que le repliement et l'oligomérisation sont deux processus concourants. Une découverte majeure est l'existence d'un équilibre monomère oligomère entre deux états I1 et I2 intermédiaires du repliement. Détecté indirectement à l'aide de RMN temps réel comme SOFAST, I2 a été directement charactérisé en SAXS: Il s'agit probablement d'un dimère. Les états intermédiaires de repliement de B2M avaient été pointés comme favorisant la formation de fibrilles: cela s'explique facilement avec l'existence d'un intermédiaire dimérique. Une combinaison de méthodes biophysiques permet la caractérisation de cet équilibre monomère-oligomère. En SAXS, puis confirmé en RMN, la stoichiométrie de l'équilibre est celle d'un monomère-dimère. Des travaux complémentaires utilisant les techniques développées pour cette étude pourront servir à caractériser plus finement cet équilibre. L'étude approfondie du repliement de B2M pousse les techniques biophysiques dans leurs retranchements: la sensibilité et le temps d'acquisition pour la RMN, la polydispersité pour le SAXS. Pourtant dans les deux cas un grand oligomère I3, qui disparait en quelques minutes, a pu être détecté, ce qui fut confirmé par UV-Fluo. La caractérisation d'I3 demandera des dévelopements méthodologiques supplémentaires, ainsi qu'un nouveau plan d'expérience. D'autres méthodes comme la spectrométrie de masse nano-ESI pourraient représenter des sources d'information utiles. S'attaquer aux limites des méthodes biophysiques pousse au développement méthodologique. Ainsi pour étudier la structure et dynamique d'I1, la méthode d'acquisition continue des données a permis l'attribution des résonnances de cette espèce qui a une demi vie de quelques dizaines de minutes. Un échange conformationnel a été découvert pour l'état I1 du mutant W60G, en développant une méthode de relaxation RMN: R2-BEST-TROSY. Les méthodes développées pour cette étude pourront servir des études sur le repliement d'autres protéines, mais aussi dans d'autres contextes où la demi-vie des objets étudiés est courte, comme dans les expérience RMN intracellulaires. Cette étude est évidemment éloignée d'une application directe dans le combat contre les maladies du mauvais repliement des protéines. Pour autant, la découverte d'états intermédiaires oligomériques souligne que l'oligomérisation et le repliement ne devraient pas être étudiés séparément, mais sont des processus liés. Les développements méthodologiques de cette étude pourront aussi être appliqués à d'autres protéines comme à d'autres contexte. Il est donc permis d'espérer que ces questionnements et développements permettront d'avancer vers une meilleure compréhension de ces maladies.

Stroke is the leading cause of long-term disability in the developed world and can have devastating effects on the health and everyday functioning of individuals. In most cases stroke is ischemic and is caused by the obstruction of blood flow due to a clot in the brain blood vessels. This initiates a cascade of events that result in tissue death and loss of behavioural function associated with the damaged region. The peri-infarct cortex is a region surrounding the infarct core that survives the ischemic event and is most susceptible to pharmacological treatments and rehabilitation. α4ß2 nicotinic acetylcholine receptor (nAChR) signalling has been implicated as a mechanism that affects cell survival and cell death in the acute response after stroke. Nicotinic receptor signalling is also involved in modulating brain excitability, which can affect neural plasticity and restoration of cortical circuits and lead to recovery of lost function after stroke. In order to elucidate the role of α4ß2 nAChRs on acute and chronic recovery after stroke, we tested two hypotheses: (1) blocking α4ß2 nAChRs triggers acute neuroprotection and (2) α4ß2 nAChRs play a role in regulating plasticity and long-term functional recovery. In the first set of experiments a new model of targeted photothrombotic stroke was induced in a distal branch of the middle cerebral artery (MCA) in awake and anaesthetized mice. Mice treated with the α4ß2 nAChR antagonist dihydro-ß-erythroidine (DHßE) showed smaller lesion sizes relative to vehicle controls and this effect was greater in mice that were awake during stroke induction. To determine the mechanism of α4ß2 nAChRmediated neuroprotection, changes in collateral flow were measured using Evans bluestained surface angiograms and laser Doppler flowmetry. Contrary to what was expected, DHßE did not appear to induce neuroprotection by altering collateral flow. In the second set of experiments, we first used confocal imaging to quantify and characterize the expression of α4ß2 nAChRs after stroke. Next, mice were induced with a targeted photothrombotic stroke in the forelimb somatosensory cortex. Mice were then chronically treated with DHßE to determine if α4ß2 nAChR antagonism could improve recovery of function. Behavioural tests showed that blocking α4ß2 nAChRs chronically had no effect on forelimb function after stroke. Voltage-sensitive dye imaging was used to measure forelimb-evoked responses in the somatosensory cortex and revealed no differences in cortical responsiveness between treated and non-treated groups. Altogether, these results show that changes in α4ß2 nAChR signalling that occur after stroke mediate ischemic cell death but do not have an effect on long-term recovery and plasticity. Moreover, they present a novel pathway for investigating stroke pathophysiology and the development of acute neuroprotective treatments. / Graduate / 0317 / aseto@uvic.ca

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Stuttgart, Univ., Diss., 2002.

Aims: The purpose of the research was to investigate the benefits of a hereditary colorectal cancer registry in the management of patients and families with Lynch syndrome. In study one, a systematic review was performed to quantify the impact of registration and screening on colorectal cancer (CRC) incidence and mortality, with comparison between familial adenomatous polyposis (FAP) and Lynch syndrome (LS). In study two, a regional Lynch syndrome registry was utilised to evaluate the uptake of predictive testing and colorectal screening among first-degree relatives (FDRs) and investigate novel methods for engaging at-risk relatives, including an enhanced role for the general practitioner (GP). In study three, the registry was used to investigate proposed associations between Lynch syndrome and prostate and bladder cancer. In study four, mismatch repair-deficient (dMMR) CRCs from Lynch syndrome patients and randomised-controlled trials (RCTs) were used to evaluate a novel prognostic biomarker, beta-2 microglobulin (B2M). Methods: An electronic database search was conducted to identify studies describing CRC incidence and/or mortality in FAP or LS, with comparison of either: 1) screened and unscreened patients or 2) patients ‘before and after’ establishment of the registry. Using the Manchester regional Lynch syndrome registry database, the uptake of predictive testing and colorectal screening among FDRs was assessed with Kaplan-Meier analysis. Novel strategies for improving engagement were explored via a patient advisory group discussion and a regional primary care questionnaire. Cases of prostate and bladder cancer in male mutation carriers and their male FDRs were identified, and cumulative and relative risks were calculated, using expected rates from cancer registry data. DNA from 350 dMMR CRC specimens from Lynch syndrome patients and RCTs were tested for B2M mutations using Sanger sequencing, and correlated with clinical outcome. Results: 43 studies were included in the systematic review (33 FAP; 10 Lynch). Registry-based screening was associated with a significant reduction in CRC incidence and in Lynch syndrome, CRC-related mortality was negligible in those undergoing surveillance. 242 Lynch syndrome families were recorded on the Manchester Lynch syndrome registry. 329 of 591 (55.7%) eligible FDRs had undergone predictive testing. Uptake was significantly lower in males and younger age groups (<25 yrs). Compliance with colorectal screening was excellent following a mutation positive predictive test but poor in untested individuals (97.3% vs 35.0%). Eight prostate cancers were identified in 821 male LS mutation carriers and male FDRs. MSH2 mutation carriers had a ten-fold increased risk of prostate cancer (RR 10.41; 95%CI 2.80, 26.65) but no association with bladder cancer was identified. 69/286 (24.1%) of dMMR CRCs contained significant B2M mutations. B2M mutations were associated with complete absence of recurrence (0/39) during follow-up in the QUASAR trial (stage II), compared with 14/77 (18.2%) in wild-type B2M (p=0.005). Conclusion: Studies consistently report that registration and screening result in a reduction of CRC incidence and mortality in FAP and LS (Level 2a evidence, Grade B recommendation). Funding and managerial support for registries should be made available. Uptake of predictive testing and colorectal screening in Lynch syndrome could be substantially improved, particularly among males and younger age groups, but this requires advances in communication with at-risk relatives. It is unlikely that GPs will actively participate without considerable support from genetics services. A trial of PSA screening in MSH2 mutation carriers from 50 years would be appropriate. B2M mutation status has potential clinical utility as a prognostic biomarker in stage II dMMR CRC.