Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes

We developed a new 2-day protocol for the generation of dendritic cells (DCs) from human monocytes in vitro. First, we demonstrated that 24 hours of culture with GM-CSF and IL-4 are sufficient to generate immature DCs capable of antigen uptake. We then compared two different strategies for DC maturation: proinflammatory mediators were either added together with GM-CSF and IL-4 from the beginning of cell culture or added after 24 hours of differentiation with GM-CSF and IL-4. After 48 hours of total culture period, expression of activation markers was more pronounced in cells generated by the 2-step differentiation and activation method. Our new protocol for 2-day DC differentiation reduces labor, cost and time and also reliably renders high numbers of mature and viable DCs

Exclusion of PINK1 as candidate gene for the late-onset form of Parkinson's disease in two European populations

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disorder. Recently, mutations in the PINK1 (PARK6) gene were shown to rarely cause autosomal-recessively transmitted, early-onset parkinsonism. In order to evaluate whether PINK1 contributes to the risk of common late-onset PD we analysed PINK1 sequence variations. A German (85 patients) and a Norwegian cohort (90 patients) suffering from late-onset PD were screened for mutations and single nucleotide polymorphisms (SNPs) in the PINK1 gene. Both cohorts consist of well-characterized patients presenting a positive family history of PD in ~17%. Investigations were performed by single strand conformation polymorphism (SSCP), denaturating high performance liquid chromatography (DHPLC) and sequencing analyses. SNP frequencies were compared by the χ(2 )test RESULTS: Several common SNPs were identified in our cohorts, including a recently identified coding variant (Q115L) in exon 1. Genotyping of the Q115L variation did not reveal significant frequency differences between patients and controls. Pathogenic mutations in the PINK1 gene were not identified, neither in the German nor in the Norwegian cohort. CONCLUSION: Sequence variation in the PINK1 gene appears to play a marginal quantitative role in the pathogenesis of the late-onset form of PD, in German and Norwegian cohorts, if at all

Elevated α-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson's disease

The presynaptic protein α-synuclein is involved in several neurodegenerative diseases, including Parkinson's disease (PD). In rare familial forms of PD, causal mutations (PARK1) as well as multiplications (PARK4) of the α-synuclein gene have been identified. In sporadic, idiopathic PD, abnormal accumulation and deposition of α-synuclein might also cause degeneration of dopaminergic midbrain neurons, the clinically most relevant neuronal population in PD. Thus, cell-specific quantification of α-synuclein expression-levels in dopaminergic neurons from idiopathic PD patients in comparison to controls would provide essential information about contributions of α-synuclein to the etiology of PD. However, a number of previous studies addressing this question at the tissue-level yielded varying results regarding α-synuclein expression. To increase specificity, we developed a cell-specific approach for mRNA quantification that also took into account the important issue of variable RNA integrities of the individual human postmortem brain samples. We demonstrate that PCR –amplicon size can confound quantitative gene-expression analysis, in particular of partly degraded RNA. By combining optimized UV-laser microdissection- and quantitative RT–PCR-techniques with suitable PCR assays, we detected significantly elevated α-synuclein mRNA levels in individual, surviving neuromelanin- and tyrosine hydroxylase-positive substantia nigra dopaminergic neurons from idiopathic PD brains compared to controls. These results strengthen the pathophysiologic role of transcriptional dysregulation of the α-synuclein gene in sporadic PD

JNK Isoforms Differentially Regulate Neurite Growth and Regeneration in Dopaminergic Neurons In Vitro

Parkinson’s disease is characterized by selective and progressive loss of midbrain DAergic neurons (MDN) in the substantia nigra and degeneration of its nigrostriatal projections. Whereas the cellular pathophysiology has been closely linked to an activation of c-Jun N-terminal kinases (JNKs) and c-Jun, the involvement of JNKs in regenerative processes of the nigrostriatal pathway is controversially discussed. In our study, we utilized a mechanical scratch lesion paradigm of midbrain DAergic neurons in vitro and studied regenerative neuritic outgrowth. After a siRNA-mediated knockdown of each of the three JNK isoforms, we found that JNKs differentially regulate neurite regeneration. Knockdown of JNK3 resulted in the most prominent neurite outgrowth impairment. This effect was attenuated again by plasmid overexpression of JNK3. We also evaluated cell survival of the affected neurons at the scratch border. JNK3 was found to be also relevant for survival of MDN which were lesioned by the scratch. Our data suggest that JNK isoforms are involved in differential regulation of cell death and regeneration in MDN depending on their neurite integrity. JNK3 appears to be required for regeneration and survival in the case of an environment permissive for regeneration. Future therapeutic approaches for the DAergic system may thus require isoform specific targeting of these kinases

Interleukin-6 receptor pathways in abdominal aortic aneurysm

We conducted a systematic review and meta-analysis of studies reporting circulating IL-6 in AAA, and new investigations of the association between a common non-synonymous functional variant (Asp358Ala) in the IL-6R gene (IL6R) and AAA, followed the analysis of the variant both in vitro and in vivo. Inflammation may play a role in the development of abdominal aortic aneurysms (AAA). Interleukin-6 (IL-6) signalling through its receptor (IL-6R) is one pathway that could be exploited pharmacologically. We investigated this using a Mendelian randomization approach

A Hierarchy of Monitoring Properties for Autonomous Systems

Monitoring capabilities play a central role in mitigating safety risks of current, but especially future autonomous aircraft systems. These future systems are likely to include complex components such as neural networks for environment perception, which pose a challenge for current verification approaches; they are considered as black-box components. To assure that these black-boxes comply to their specification, they are typically monitored to detect violations during execution in respect to their input and output behavior. Such behavioral properties often include more complex aspects such as temporal or spatial notions. Besides monitoring their behavior, the outputs can also be compared to data from other assured sensors or components of the aircraft, making monitoring an even more integral part of the system, which ideally has access to all available resources to assess the overall health of the operation. Current approaches using handwritten code for monitoring functions run the risk of not being able to keep up with these challenges. Therefore, in this paper, we present a hierarchy of monitoring properties that provides a perspective for overall health. We also present a categorization of monitoring properties and show how different monitoring specification languages can be used for formalization. These monitoring languages represent a higher abstraction of general-purpose code and are therefore more compact and easier for a user to write and read. They improve the maintainability of monitoring properties that is required to handle the increased complexity of future autonomous aircraft systems

A Hierarchy of Monitoring Properties for Autonomous Systems

Monitoring capabilities play a central role in mitigating safety risks of current, and especially future autonomous aircraft systems. These future systems are likely to include complex components such as neural networks for environment perception, which pose a challenge for current verification approaches; they are considered as black-box components. To assure that these black-boxes comply with their specification, they must be monitored to detect violations during execution with respect to their input and output behaviors. Such behavioral properties often include more complex aspects such as temporal or spatial notions. The outputs can also be compared to data from other assured sensors or components of the aircraft, making monitoring an integral part of the system, which ideally has access to all available resources to assess the overall health of the operation. Current approaches using handwritten code for monitoring functions run the risk of not being able to keep up with these challenges. Therefore, in this paper, we present a hierarchy of monitoring properties that provides a perspective for overall health. We also present a categorization of monitoring properties and show how different monitoring specification languages can be used for formalization. These monitoring languages represent a higher abstraction of general-purpose code and are therefore more compact and easier for a user to write and read, and we can validate their implementations independently from the systems they reason about. They improve the maintainability of monitoring properties that is required to handle the increased complexity of future autonomous aircraft systems.This is a pre-print of the article Schirmer, Sebastian, Christoph Torens, Johann C. Dauer, Jan Baumeister, Bernd Finkbeiner, and Kristin Y. Rozier. "A Hierarchy of Monitoring Properties for Autonomous Systems." Copyright 2022 The Authors. Posted with permission