Peritoneal fluid cytokines and the relationship with endometriosis and pain*

It is generally accepted that the current scoring system for endometriosis has little correlation with clinical symptoms such as pain, and therefore we may deduce that either endometriosis does not cause pain, or that the current scoring system does not indicate the biological activity of the disease. Pain may occur because the presence of endometriosis produces an intraperitoneal inflammatory response, and several studies have shown that the cytokine content of peritoneal fluid differs between women with and without endometriosis. We studied the relationship between tumour necrosis factor a (TNFa), platelet-derived growth factor (PDGF), interleukin (IL)-6, IL-4 and TNF (a and P) activity in peritoneal fluid and the clinical history of pain and infertility. TNFa concentrations were increased in peritoneal fluid of women with endometriosis and of infertile women; PDGF concentrations were increased in peritoneal fluid of parous women; EL-6 was increased in peritoneal fluid of women with adhesions; IL-4 was absent from peritoneal fluid. PDGF and IL-6 concentrations were cycle related, with the highest amounts in the menstrual and proliferative phases respectively. We failed to demonstrate any association between concentrations of cytokines in vitro and pain symptoms or severity of endometriosis

Development of a protein microarray platform for the multiplex analysis of biomarkers associated with rheumatoid arthritis

Currently in the drug development process there is a growing awareness of the need to utilise a biomarker strategy which would allow compounds to be developed in a more efficient way with improved safety and pharmacology. Technologies which can evaluate, validate and monitor biomarkers in a cost effective and efficient manner are a necessity if such a biomarker strategy is to be properly implemented. In this thesis the development, validation and implementation of a protein microarray for quantitative and simultaneous analysis of proteins is described. In order to demonstrate the feasibility of this approach, Rheumatoid arthritis (RA) was chosen as a model for proof of concept. Based on the current literature, seven proteins thought to be associated with the development and progression of RA were selected. Initially, a protein microarray was developed on a glass chip treated either with a self assembled monolayer (SAM) of octadecyl phosphoric acid ester (ODP) or with polyL-lysine. SAM showed its superiority over poly-L-lysine by generating more homogenous and less variable spots. However, the process of coating the chip with the SAM was time consuming and expensive. Moreover, assay processing was entirely performed manually and could not be automated without a significant investment of time and resources. As a result, high inter-chip variability was observed preventing sensitive, quantitative and reproducible analysis to be performed. An attempt was, therefore, undertaken to develop an alternative microarray platform. The appearance on the market of long neck tips for antibody printing devices, provided the option of using established polystyrene 96-well plates as the solid support for developing a fully automated microarray format. The development process involved reagent selection, printing protocol optimization, matrix investigation, assay protocol establishment, and detection system evaluation. The robustness and reproducibility of the methodology was investigated using the Food and Drug Administration (FDA) regulatory guidelines for pharmacokinetic assay validation, in which a spike-recovery validation test was elaborated and run overdays. The method was shown to be both quantitative and reproducible, with an assay accuracy between 70-130%, and assay precision less than 30%. Importantly, the working range for each assay covered the relevant physiological concentrations. In addition, protein microarray performance was compared with the classical ELISA approach. Sera collected from a total of 78 individuals representing either rheumatic or healthy patients were measured using both approaches. Correlation coefficients (R2) between the two technologies was calculated for each analyte giving: 0.90 for A, 0.60 for B, 0.93 for C, 0.96 for D, 0.94 for E and 0.95 for F. Finally, the developed protein microarray was used to compare the analyte concentration levels between patients with RA and other rheumatic diseases. Significant differences in the serum concentration of B (p<0.0022), C (p<0.0107), E (p<0.0024) and F (p<0.0057) between RA and other arthritic patients were observed. In conclusion, the obtained results demonstrate the applicability of the developed protein microarray for quantitative and simultaneous analysis of the selected RA-related proteins in clinical samples. It is anticipated that miniaturized and multiplexed immunoassays which allow for the rapid evaluation of multiple analytes in a single sample, will represent a valuable tool for validating and monitoring biomarkers in the drug development process

Novel botanical drug DA-9803 prevents deficits in Alzheimer’s mouse models

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by deposition of amyloid plaques and disruption of neural circuitry, leading to cognitive decline. Animal models of AD deposit senile plaques and exhibit structural and functional deficits in neurons and neural networks. An effective treatment would prevent or restore these deficits, including calcium dyshomeostasis observed with in-vivo imaging. Methods: We examined the effects of DA-9803, a multimodal botanical drug, in 5XFAD and APP/PS1 transgenic mice which underwent daily oral treatment with 30 or 100 mg/kg DA-9803 or vehicle alone. Behavioral testing and longitudinal imaging of amyloid deposits and intracellular calcium in neurons with multiphoton microscopy was performed. Results: Chronic administration of DA-9803 restored behavioral deficits in 5XFAD mice and reduced amyloid-β levels. DA-9803 also prevented progressive amyloid plaque deposition in APP/PS1 mice. Elevated calcium, detected in a subset of neurons before the treatment, was restored and served as a functional indicator of treatment efficacy in addition to the behavioral readout. In contrast, mice treated with vehicle alone continued to progressively accumulate amyloid plaques and calcium overload. Conclusions: In summary, treatment with DA-9803 prevented structural and functional outcome measures in mouse models of AD. Thus, DA-9803 shows promise as a novel therapeutic approach for Alzheimer’s disease. Electronic supplementary material The online version of this article (10.1186/s13195-018-0338-2) contains supplementary material, which is available to authorized users

Brain distribution and release of Cholecystokinin octapeptide

In this thesis the in vitro release of immunoreactive CCK₈ (iCCK₈) from rat central nervous system preparations and the regulation of this release have been studied. Rat brain was dissected into the following regions; hypothalamus, cerebral cortex, striatum and thalamus, according to the method of Brownstein, Arimura, Sato et. al. (1975). CCK₈ was found to be distributed throughout these regions (range of 9 - 300 pmoles), with the highest concentration in cortex (300 pmol). In addition, low levels (range 9 - 30 pmoles) of CCK₈ were found in spinal cord, brain stem and cerebellum, in agreement with other workers. Immunohistochemical techniques have demonstrated CCK-like immunoreactivity in nerve cell bodies and fibres throughout brain, particularly in the cortex. Subcellular fractionation of rat brain was used to study the subcellular localisation of CCK. Tissue was homogenised to shear off nerve terminals (synaptosomes) which were purified and used to study the release of the peptide from hypothalamic and extrahypothalamic nerve endings

Beurteilung des Knochenstoffwechsels bei Patienten mit chronischer Herzinsuffizienz - 1-Alfacalcidol als Prävention und Therapie der sekundären Osteoporose

Fortschreitender Knochenmasseverlust und sekundäre Osteoporose sind häufige Komplikationen bei Patienten mit chronischer Herzinsuffizienz. Aufgrund von Schmerzen, spontanen Frakturen und eventuell dauerhafter Immobilisation werden die ohnehin schon schwer erkrankten Patienten noch weiter beeinträchtigt. Bisher jedoch gibt es noch keine Standardtherapie für die sekundäre Osteoporose nach Herzinsuffizienz. In vorliegender Arbeit wurde deshalb erstmals der Effekt einer Osteoporose-Therapie mit Alfacalcidol (1-α-Hydroxy-Vitamin D3) plus Calcium auf den Knochenstoffwechsel von Patienten mit chronischer Herzinsuffizienz untersucht. Dies geschah in interdisziplinärer Zusammenarbeit mit der Medizinischen Poliklinik Innenstadt der Ludwig-Maximilians-Universität München. Es sollte evaluiert werden, ob diese Therapie entscheidende Vorteile gegenüber der alleinigen prophylaktischen Gabe von Calcium bietet. Patienten mit chronischer Herzinsuffizienz erhielten im ersten Studienjahr eine Basisgabe von 500 mg Calcium und im 2. Studienjahr eine Kombinationstherapie aus 1 µg Alfacalcidol plus 500 mg Calci-um. Als Hauptzielgrößen für den Therapieerfolg dienten die biochemischen Parameter des Knochenstoffwechsels und die Entwicklung der Knochendichte. Als Marker der Knochenformation wurden das nichtkollagene Knochenprotein Osteocalcin und die Knochenspezifische Alkalische Phosphatase, als Marker der Knochenresorption die Pyridinium-Crosslinks Pyridinolin und Desoxypyridinolin herangezogen. Die Messung der Knochendichte erfolgte mittels Zweispektren-Röntgenabsorptiometrie (DXA) an Lendenwirbelsäule (LWS) und Femur. Zu Studienbeginn zeigten die Patienten einen gestörten Knochenstoffwechsel mit physiologischer Knochenformation und erhöhter Knochenresorption. Die mittlere Knochendichte an Femur und Len-denwirbelsäule war gemäß der WHO-Definition im Sinne einer Osteopenie vermindert. Eine Osteopo-rose war bei 18 % der Studienteilnehmer am Femur und bei 23 % an der LWS nachweisbar. 41 % der Patienten litten zudem an einem sekundären Hyperparathyreoidismus, welcher einen wichtigen Faktor bei der Krankheitsentstehung darstellt. Die Untersuchung ergab, dass unter dem Einfluss der alleinigen Calciumgabe im ersten Studienjahr die Knochenresorption weiter anstieg und auch der fortschreitende Verlust an Knochenmasse nicht aufgehalten werden konnte. Hingegen bewirkte die Therapie mit Alfacalcidol plus Calcium im zweiten Studienjahr eine Normalisierung des Knochenstoffwechsels. Die Knochendichte an der Lendenwirbelsäule und am Femurhals stieg unter der Therapie mit Alfacalcidol plus Calcium hochsignifikant an. In vorliegender Arbeit konnte gezeigt werden, dass die alleinige Gabe von Calcium nicht geeignet ist, den pathologischen Knochenstoffwechselvorgängen bei Patienten mit chronischer Herzinsuffizienz entgegen zu wirken. Hingegen stellt die Kombination aus Alfacalcidol plus Calcium eine geeignete Therapie dar, durch welche die erhöhte Knochenresorption gehemmt, der fortschreitende Knochenverlust aufgehalten und in Folge eine Steigerung der Knochenmasse herbeigeführt wird