Andreas Kulawik

Tissue macrophages suppress viral replication and prevent severe immunopathology in an interferon-I-dependent manner in mice

The innate immune response plays an essential role in the prevention of early viral dissemination. We used the lymphocytic choriomeningitis virus model system to analyze the role of tissue macrophages/Kupffer cells in this process. Our findings demonstrated that Kupffer cells are essential for the efficient capture of infectious virus and for preventing viral replication. The latter process involved activation of Kupffer cells by interferon (IFN)‐I and prevented viral spread to neighboring hepatocytes. In the absence of Kupffer cells, hepatocytes were not able to suppress virus replication, even in the presence of IFN‐I, leading to prolonged viral replication and severe T cell‐dependent immunopathology. Conclusion: Tissue‐resident macrophages play a crucial role in early viral capture and represent the major liver cell type exhibiting responsiveness to IFN‐I and providing control of viral replication. (HEPATOLOGY 2010)

Single Fibril Growth Kinetics of α-Synuclein

Neurodegenerative disorders associated with protein misfolding are fatal diseases that are caused by fibrillation of endogenous proteins such as α-synuclein (α-syn) in Parkinsons disease (PD) or amyloid-β in Alzheimers disease. Fibrils of α-syn are a major pathological hallmark of PD and certain aggregation intermediates are postulated to cause synaptic failure and cell death of dopaminergic neurons in the substantia nigra. For the development of therapeutic approaches, the mechanistic understanding of the fibrillation process is essential. Here we report real-time observation of α-syn fibril elongation on a glass surface, imaged by total internal reflection fluorescence microscopy using thioflavin T fluorescence. Fibrillation on the glass surface occurred in the same time frame and yielded fibrils of similar length as fibrillation in solution. Time-resolved imaging of fibrillation on a single fibril level indicated that α-syn fibril elongation follows a stop-and-go mechanism; that is, fibrils either extend at a homogenous growth rate or stop to grow for variable time intervals. The fibril growth kinetics were compatible with a model featuring two states, a growth state and a stop state, which were approximately isoenergetic and interconverted with rate constants of ~1.5×10(-4) s(-1). In the growth state, α-syn monomers were incorporated into the fibril with a rate constant of 8.6×10(3) M(-1) s(-1). Fibril elongation of α-syn is slow compared to other amyloidogenic proteins.

Application of an Amyloid Beta Oligomer Standard in the sFIDA Assay

Still, there is need for significant improvements in reliable and accurate diagnosis for Alzheimers disease (AD) at early stages. It is widely accepted that changes in the concentration and conformation of amyloid-β (Aβ) appear several years before the onset of first symptoms of cognitive impairment in AD patients. Because Aβ oligomers are possibly the major toxic species in AD, they are a promising biomarker candidate for the early diagnosis of the disease. To date, a variety of oligomer-specific assays have been developed, many of them ELISAs. Here, we demonstrate the sFIDA assay, a technology highly specific for Aβ oligomers developed toward single particle sensitivity. By spiking stabilized Aβ oligomers to buffer and to body fluids from control donors, we show that the sFIDA readout correlates with the applied concentration of stabilized oligomers diluted in buffer, cerebrospinal fluid (CSF), and blood plasma over several orders of magnitude. The lower limit of detection was calculated to be 22 fM of stabilized oligomers diluted in PBS, 18 fM in CSF, and 14 fM in blood plasma.

Biofunctionalized Silica Nanoparticles: Standards in Amyloid-β Oligomer-Based Diagnosis of Alzheimer’s Disease

Amyloid-β (Aβ) oligomers represent a promising biomarker for the early diagnosis of Alzheimers disease (AD). However, state-of-the-art methods for immunodetection of Aβ oligomers in body fluids show a large variability and lack a reliable and stable standard that enables the reproducible quantitation of Aβ oligomers. At present, the only available standard applied in these assays is based on a random aggregation process of synthetic Aβ and has neither a defined size nor a known number of epitopes. In this report, we generated a highly stable standard in the size range of native Aβ oligomers that exposes a defined number of epitopes. The standard consists of a silica nanoparticle (SiNaP), which is functionalized with Aβ peptides on its surface (Aβ-SiNaP). The different steps of Aβ-SiNaP synthesis were followed by microscopic, spectroscopic and biochemical analyses. To investigate the performance of Aβ-SiNaPs as an appropriate standard in Aβ oligomer immunodetection, Aβ-SiNaPs were diluted in cerebrospinal fluid and quantified down to a concentration of 10 fM in the sFIDA (surface-based fluorescence intensity distribution analysis) assay. This detection limit corresponds to an Aβ concentration of 1.9 ng l-1 and lies in the sensitivity range of currently applied diagnostic tools based on Aβ oligomer quantitation. Thus, we developed a highly stable and well-characterized standard for the application in Aβ oligomer immunodetection assays that finally allows the reproducible quantitation of Aβ oligomers down to single molecule level and provides a fundamental improvement for the worldwide standardization process of diagnostic methods in AD research.

Prenylated flavanone derivatives isolated from Erythrina addisoniae are potent inducers of apoptotic cell death

Extracts of Erythrina addisoniae are frequently used in the traditional medicine of Western Africa, but insufficient information about active compounds is available. From the stem bark of E. addisoniae, three (1, 2, 4) and three known (3, 5, 6) flavanones were isolated: addisoniaflavanones I and II, containing either a 2″,3″-epoxyprenyl moiety (1) or a 2″,3″-dihydroxyprenyl moiety (2) were shown to be highly toxic (MTT assay: EC50 values of 5.25±0.7 and 8.5±1.3 μM, respectively) to H4IIE hepatoma cells. The cytotoxic potential of the other isolated flavanones was weaker (range of EC50 values between 15 and >100 μM). Toxic effects of addisoniaflavanone I and II were detectable after 3h (MTT assay). Both compounds induced an apoptotic cell death (caspase-3/7 activation, nuclear fragmentation) in the hepatoma cells and, at high concentrations, also necrosis (membrane disruption: ethidium bromide staining). Formation of DNA strand breaks was not detectable after incubation with these compounds (comet assay). In conclusion, the prenylated flavanones addisoniaflavanones I and II may be of interest for pharmacological purposes due to their high cytotoxic and pro-apoptotic potential against hepatoma cells.

Analysis of anticoagulants for blood-based quantitation of amyloid β oligomers in the sFIDA assay

Abstract Early diagnostics at the preclinical stage of Alzheimer’s disease is of utmost importance for drug development in clinical trials and prognostic guidance. Since soluble Aβ oligomers are considered to play a crucial role in the disease pathogenesis, several methods aim to quantify Aβ oligomers in body fluids such as cerebrospinal fluid (CSF) and blood plasma. The highly specific and sensitive method surface-based fluorescence intensity distribution analysis (sFIDA) has successfully been established for oligomer quantitation in CSF samples. In our study, we explored the sFIDA method for quantitative measurements of synthetic Aβ particles in blood plasma. For this purpose, EDTA-, citrate- and heparin-treated blood plasma samples from five individual donors were spiked with Aβ coated silica nanoparticles (Aβ-SiNaPs) and were applied to the sFIDA assay. Based on the assay parameters linearity, coefficient of variation and limit of detection, we found that EDTA plasma yields the most suitable parameter values for quantitation of Aβ oligomers in sFIDA assay with a limit of detection of 16 fM.

Nanoparticle standards for immuno-based quantitation of α-synuclein oligomers in diagnostics of Parkinson's disease and other synucleinopathies

Parkinsons disease (PD) is a neurodegenerative disorder that is characterized by symptoms such as rigor, tremor and bradykinesia. A reliable and early diagnosis could improve the development of early therapeutic strategies before death of dopaminergic neurons leads to the first clinical symptoms. The sFIDA (surface-based fluorescence intensity distribution analysis) assay is a highly sensitive method to determine the concentration of α-synuclein (α-syn) oligomers which are presumably the major toxic isoform of α-syn and potentially the most direct biomarker for PD. Oligomer-based diagnostic tests require standard molecules that closely mimic the native oligomer. This is particularly important for calibration and assessment of inter-assay variation. In this study, we generated a standard in form of α-syn coated silica nanoparticles (α-syn-SiNaPs) that are in the size range of α-syn oligomers and provide a defined number of α-syn epitopes. The preparation of the sFIDA assay was realized on an automated platform to allow handling of high number of samples and reduce the effects of human error. The assay outcome was analyzed by determination of coefficient of variation and linearity for the applied α-syn-SiNaPs concentrations. Additionally, the limit of detection and lower limit of quantification were determined yielding concentrations in the lower femtomolar range.

IL-1-induced and p38MAPK-dependent activation of the mitogen-Activated protein kinase-Activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

The IL-1β induced activation of the p38MAPK/MAPK-activated protein kinase 2 (MK2) pathway in hepatocytes is important for control of the acute phase response and regulation of liver regeneration. Many aspects of the regulatory relevance of this pathway have been investigated in immune cells in the context of inflammation. However, very little is known about concentration-dependent activation kinetics and signal propagation in hepatocytes and the role of MK2. We established a mathematical model for IL-1β-induced activation of the p38MAPK/MK2 pathway in hepatocytes that was calibrated to quantitative data on time- and IL-1β concentration-dependent phosphorylation of p38MAPK and MK2 in primary mouse hepatocytes. This analysis showed that, in hepatocytes, signal transduction from IL-1β via p38MAPK to MK2 is characterized by strong signal amplification. Quantification of p38MAPK and MK2 revealed that, in hepatocytes, at maximum, 11.3% of p38MAPK molecules and 36.5% of MK2 molecules are activated in response to IL-1β. The mathematical model was experimentally validated by employing phosphatase inhibitors and the p38MAPK inhibitor SB203580. Model simulations predicted an IC50 of 1–1.2 μm for SB203580 in hepatocytes. In silico analyses and experimental validation demonstrated that the kinase activity of p38MAPK determines signal amplitude, whereas phosphatase activity affects both signal amplitude and duration. p38MAPK and MK2 concentrations and responsiveness toward IL-1β were quantitatively compared between hepatocytes and macrophages. In macrophages, the absolute p38MAPK and MK2 concentration was significantly higher. Finally, in line with experimental observations, the mathematical model predicted a significantly higher half-maximal effective concentration for IL-1β-induced pathway activation in macrophages compared with hepatocytes, underscoring the importance of cell type-specific differences in pathway regulation.

Advancements of the sFIDA method for oligomer‐based diagnostics of neurodegenerative diseases

Early diagnosis of Alzheimers disease (AD) is of great importance for the development of therapeutics and their application in the clinical environment. Amyloid β (Aβ) oligomers are crucial for the onset and progression of AD and represent a popular drug target, being presumably the most direct biomarker. Efforts to measure Aβ oligomers in body fluids are hampered by the low analyte concentration and presence of Aβ monomers. The surface‐based fluorescence intensity distribution analysis (sFIDA) features both highly specific and sensitive oligomer quantitation as well as total insensitivity towards monomers. In this Review, we highlight structural features of oligomeric and fibrillar Aβ. Recent advancements in sFIDA assay development have been the successful automation, adaption for additional biomarkers such as α‐synuclein oligomers, and significant improvement of essential assay parameters.

SFIDA: PLATFORM TECHNOLOGY FOR DIAGNOSTICS OF PROTEIN MISFOLDING DISEASES

monomers in a solution of the same formula used for the oligomers. Polyclonal antiAbx-40 antibodies were produced in our lab. PC12 cells were grown overnight in 96-well plates at 3 x 10 cells/well, and then incubated with (a) Ab1-40 oligomers, (b) Ab1-40 monomers, (c) Ab1-40 oligomers + anti-Abx-40 antibodies, (d) Ab1-40 monomers + anti-Abx-40 antibodies, or (e) vehicle. The different treatments were assayed at different concentrations and co-cultured for an additional 24 hours. To evaluate Ab1-40-mediated cytotoxicity, PC12 cell viability was quantified by using the MTT assay (colorimetric assay for assessing cell metabolic activity). Results:Ab1-40 oligomers produced a concentration-dependent cytotoxic effect on PC12 cells whereas Ab1-40 monomers did not. Pre-incubation of Ab1-40 monomers with anti-Abx-40 antibodies prevented the formation of Ab1-40 oligomers, resulting in increased cell viability in the cultures. The beneficial effect of anti-Abx-40 antibodies was dependent on the extent of oligomerization, being more protective when present at the beginning of oligomer formation than once the oligomers have been formed. Conclusions: This study shows that antibodies against the C-terminal end of Ab1-40 protect from amyloid-beta cytotoxicity by preventing the formation of oligomers. Thus, active immunization with ABvac40 appears to be a promising preventive approach for the treatment of AD.