Tissue-resident macrophages are key players in inflammatory processes, and their activation and functionality are crucial in health and disease. Numerous diseases are associated with alterations in ...homeostasis or dysregulation of the innate immune system, including allergic reactions, autoimmune diseases, and cancer. Macrophages are a prime target for drug discovery due to their major regulatory role in health and disease. Currently, the main sources of macrophages used for therapeutic compound screening are primary cells isolated from blood or tissue or immortalized or neoplastic cell lines (e.g., THP-1). Here, we describe an improved method to employ induced pluripotent stem cells (iPSCs) for the high-yield, large-scale production of cells resembling tissue-resident macrophages. For this, iPSC-derived macrophage-like cells are thoroughly characterized to confirm their cell identity and thus their suitability for drug screening purposes. These iPSC-derived macrophages show strong cellular identity with primary macrophages and recapitulate key functional characteristics, including cytokine release, phagocytosis, and chemotaxis. Furthermore, we demonstrate that genetic modifications can be readily introduced at the macrophage-like progenitor stage in order to interrogate drug target-relevant pathways. In summary, this novel method overcomes previous shortcomings with primary and leukemic cells and facilitates large-scale production of genetically modified iPSC-derived macrophages for drug screening applications.
Synucleinopathies including Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal α-synuclein in intraneuronal inclusions, named Lewy bodies. ...Mutations in GBA1, the gene encoding the lysosomal hydrolase glucocerebrosidase, have been identified as the most common genetic risk factor for PD and DLB. However, despite extensive research, the mechanism by which glucocerebrosidase dysfunction increases the risk for PD or DLB still remains elusive.
In our study we expand the toolbox for PD-DLB post-mortem studies by introducing new quantitative biochemical assays for glucocerebrosidase and α-synuclein. Applying causal modelling, we determine how these parameters are interrelated and ultimately impact disease manifestation.
We developed quantitative immuno-based assays for glucocerebrosidase and α-synuclein (total and phosphorylated at Serine 129) protein levels, as well as a liquid chromatography–mass spectrometry method for the detection of the glucocerebrosidase lipid substrate glucosylsphingosine. These assays were applied on tissue samples from frontal cortex, putamen and substantia nigra of PD (n = 15) and DLB (n = 15) patients and age-matched non-demented controls (n = 15).
Our results confirm elevated p-129 over total α-synuclein levels in the insoluble fraction of PD and DLB post-mortem brain tissue and we found significantly increased α-synuclein levels in the soluble fractions in PD and DLB. Furthermore, we identified an inverse correlation between reduced glucocerebrosidase enzyme activity and protein levels with increased glucosylsphingosine levels. In the substantia nigra, a brain region particularly vulnerable in Parkinson's disease, we found a significant correlation between glucocerebrosidase protein reduction and increased p129/total α-synuclein ratios.
We assessed the direction and strength of the interrelation between all measured parameters by confirmatory path analysis. Interestingly, we found that glucocerebrosidase dysfunction impacts the PD-DLB status by increasing α-synuclein ratios in the substantia nigra, which was partly mediated by increasing glucosylsphingosine levels.
In conclusion, we show that the introduced immuno-based assays enable the quantitative assessment of glucocerebrosidase and α-synuclein parameters in post-mortem brain. In the substantia nigra, reduced glucocerebrosidase levels contribute to the increase in α-synuclein levels and to PD-DLB disease manifestation partly by increasing its glycolipid substrate glucosylsphingosine. This interrelation between glucocerebrosidase, glucosylsphingosine and α-synuclein parameters supports the hypothesis that glucocerebrosidase acts as a modulator of PD-DLB.
•Path analysis - a new tool to understand the GBA/ a-syn axis•Ser129 phosphorylated a-syn levels are increased in LBD•In the SN reduced glucocerebrosidase protein correlates with increased α-synuclein•Glucocerebrosidase contributes to LBD partly by increasing glucosylsphingosine
A major obstacle in diabetes is the metabolic or hyperglycemic memory, which lacks specific therapies. Here we show that glucose-mediated changes in gene expression largely persist in diabetic kidney ...disease (DKD) despite reversing hyperglycemia. The senescence-associated cyclin-dependent kinase inhibitor p21 (Cdkn1a) was the top hit among genes persistently induced by hyperglycemia and was associated with induction of the p53-p21 pathway. Persistent p21 induction was confirmed in various animal models, human samples and in vitro models. Tubular and urinary p21-levels were associated with DKD severity and remained elevated despite improved blood glucose levels in humans. Mechanistically, sustained tubular p21 expression in DKD is linked to demethylation of its promoter and reduced DNMT1 expression. Two disease resolving agents, protease activated protein C (3K3A-aPC) and parmodulin-2, reversed sustained tubular p21 expression, tubular senescence, and DKD. Thus, p21-dependent tubular senescence is a pathway contributing to the hyperglycemic memory, which can be therapeutically targeted.
Introduction Spontaneous cervical artery dissection (sCAD) is a rare vasculopathy whose trigger is still unknown. We hypothesized that autoimmunity against components of the vascular wall might play ...a critical role in sCAD and examined anti-collagen type I antibodies in patients with sCAD, acute ischemic stroke, patients with thromboendarterectomy, and controls. Methods Fifty-seven patients with sCAD (age 45.7 ± 10.2 years, female 18 (31.6%)) were prospectively enrolled in four German stroke centers. Blood samples were collected at baseline, at day 10 ± 3, and after 6 ± 1 months. Patients with ischemic stroke not related to CAD (n=54, age 56.7 ± 13.7 years, female 15 (27.8%)), healthy probands (n=80, age 57.4 ± 12.9 years, female 56 (70%)), and patients undergoing thromboendarterectomy of the carotid artery (n=9, age 70.7 ± 9.3 years, female 2 (22.2%)) served as controls. Anti-collagen type I antibodies were determined by enzyme-linked immunosorbent assays (ELISAs). Results Patients with acute sCAD had higher serum levels of anti-collagen type I antibodies (33.9 ± 24.6 µg/ml) than probands (18.5 ± 11.0 µg/ml; p <0.001) but lower levels than patients with ischemic stroke not related to sCAD (47.8 ± 28.4 µg/ml; p=0.003). In patients with sCAD, serum levels of anti-collagen type I antibodies were similar in the acute, subacute, and chronic phase. Levels of anti-collagen type I antibodies significantly correlated with circulating collagen type I (rho=0.207, p=0.003). Conclusion Anti-collagen type I antibodies seem not to represent a trigger for acute sCAD or ischemic stroke but may rather be linked to the metabolism and turnover of collagen type I.
Diabetes mellitus is a metabolic disease largely due to lifestyle and nutritional imbalance, resulting in insulin resistance, hyperglycemia and vascular complications. Diabetic kidney disease (DKD) ...is a major cause of end-stage renal failure contributing to morbidity and mortality worldwide. Therapeutic options to prevent or reverse DKD progression are limited. Endothelial and glomerular filtration barrier (GFB) dysfunction and sterile inflammation are associated with DKD. Neutrophil extracellular traps (NETs), originally identified as an innate immune mechanism to combat infection, have been implicated in sterile inflammatory responses in non-communicable diseases. However, the contribution of NETs in DKD remains unknown. Here, we show that biomarkers of NETs are increased in diabetic mice and diabetic patients and that these changes correlate with DKD severity. Mechanistically, NETs promote NLRP3 inflammasome activation and glomerular endothelial dysfunction under high glucose stress in vitro and in vivo. Inhibition of NETs (PAD4 inhibitor) ameliorate endothelial dysfunction and renal injury in DKD. Taken together, NET-induced sterile inflammation promotes diabetes-associated endothelial dysfunction, identifying a new pathomechanism contributing to DKD. Inhibition of NETs may be a promising therapeutic strategy in DKD.
Viral meningitis/encephalitis (ME) is a rare but potentially harmful disease. The prompt identification of the respective virus is important to guide not only treatment but also potential public ...health countermeasures. However, in about 40% of cases, no virus is identified despite an extensive diagnostic workup. The aim of the present study was to analyze demographic, seasonal, and routine cerebrospinal fluid (CSF) parameters in cases of viral ME and assess their utility for the prediction of the causative virus.
Demographic data, season, and routine CSF parameters (total leucocytes, CSF cell differentiation, age-adjusted CSF/serum albumin ratio, and total immunoglobulin ratios) were retrospectively assessed in cases of viral ME.
In total, 156 cases of acute viral ME (74 female, median age 40.0 years) were treated at a tertiary-care hospital in Germany. Specific viral infections were detected in 93 (59.6%) cases. Of these, 14 (9.0%) cases were caused by herpes simplex virus (HSV), 36 (23.1%) by varicella-zoster virus (VZV), 27 (17.3%) by enteroviruses, 9 (5.8%) by West Nile virus (WNV), and 7 (4.5%) by other specific viruses. Additionally, 64 (41.0%) cases of ME of unknown viral etiology were diagnosed. Cases of WNV ME were older, predominantly male, showed a severe disruption of the blood-CSF-barrier, a high proportion of neutrophils in CSF, and an intrathecal total immunoglobulin M synthesis in the first CSF sample. In a multinominal logistic regression analysis, the accuracy of these CSF parameters together with age and seasonality was best for the prediction of WNV (87.5%), followed by unknown viral etiology (66.7%), VZV (61.8%), and enteroviruses (51.9%).
Cases with WNV ME showed a specific pattern of routine CSF parameters and demographic data that allowed for their identification with good accuracy. These findings might help to guide the diagnostic workup in cases with viral ME, in particular allowing the timely identification of cases with ME due to WNV.
Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by motor and non-motor symptoms. There is increasing evidence that PD pathology is accompanied by an inflammatory ...response. This is highly relevant for understanding disease progression and the development of novel neuroprotective therapies.
Assessing potential dysregulation of a panel of inflammatory mediators in the peripheral blood mononuclear cells (PBMCs) and plasma of PD patients and in the context of clinical outcome metrics.
We performed a screening of selected cell-surface chemokine receptors and adhesion molecules in PBMCs from PD patients and age-matched healthy controls in a flow cytometry-based assay. ELISA was used to quantify VCAM1 levels in the plasma of PD patients. Lymphocytic chemotactic ability was assessed using a modified Boyden chamber assay.
VLA4 expression was significantly downregulated on CD3+ T cells, CD56+ NK cells, and CD3+/CD56+ NK-T cells from PD patients; further, an increase of the soluble VLA4 ligand VCAM1 in patient plasma was noted. sVCAM1 in PD patients was even higher than reported for patients with multiple sclerosis, neuromyelitis optica, and rheumatoid arthritis. sVCAM1 levels correlated with the disease stage (Hoehn and Yahr scale) and motor impairment. Chemoattraction with SDF-1α revealed impaired motility of lymphocytes from PD patients relative to controls.
Our data provides evidence for a functional dysregulation of the sVCAM1-VLA4 axis in PD. Further studies evaluating the therapeutic potential of this axis are warranted.
Plant sterols (PSs) cannot be synthesized in mammals and are exclusively diet-derived. PSs cross the blood-brain barrier and may have anti-neuroinflammatory effects. Obesity is linked to lower ...intestinal uptake and blood levels of PSs, but its effects in terms of neuroinflammation-if any-remain unknown. We investigated the effect of high-fat diet-induced obesity on PSs in the brain and the effects of the PSs campesterol and β-sitosterol on in vitro microglia activation. Sterols (cholesterol, precursors, PSs) and polyunsaturated fatty acid-derived lipid mediators were measured in the food, blood, liver and brain of C57BL/6J mice. Under a PSs-poor high-fat diet, PSs levels decreased in the blood, liver and brain (>50%). This effect was reversible after 2 weeks upon changing back to a chow diet. Inflammatory thromboxane B2 and prostaglandin D2 were inversely correlated to campesterol and β-sitosterol levels in all brain regions. PSs content was determined post mortem in human cortex samples as well. In vitro, PSs accumulate in lipid rafts isolated from SIM-A9 microglia cell membranes. In summary, PSs levels in the blood, liver and brain were associated directly with PSs food content and inversely with BMI. PSs dampen pro-inflammatory lipid mediators in the brain. The identification of PSs in the human cortex in comparable concentration ranges implies the relevance of our findings for humans.
It is controversial whether lifestyle-induced weight loss (LIWL) intervention provides long-term benefit. Here, we investigated whether the degree of weight loss (WL) in a controlled LIWL ...intervention study determined the risk of prediabetes and recurrence of metabolic syndrome (MetS) during a 5-year follow-up. Following LIWL, 58 male participants (age 45−55 years) were divided into four quartiles based on initial WL: Q1 (WL 0−8.1%, n = 15), Q2 (WL 8.1−12.8%, n = 14), Q3 (WL 12.8−16.0%, n = 14), and Q4 (WL 16.0−27.5%, n = 15). We analyzed changes in BMI, HDL cholesterol, triglycerides (TGs), blood pressure, and fasting plasma glucose (FPG) at annual follow-up visits. With a weight gain after LIWL between 1.2 (Q2) and 2.5 kg/year (Q4), the reduction in BMI was maintained for 4 (Q2, p = 0.03) or 5 (Q3, p = 0.03; Q4, p < 0.01) years, respectively, and an increase in FPG levels above baseline values was prevented in Q2−Q4. Accordingly, there was no increase in prediabetes incidence after LIWL in participants in Q2 (up to 2 years), Q3 and Q4 (up to 5 years). A sustained reduction in MetS was maintained in Q4 during the 5-year follow-up. The present data indicate that a greater initial LIWL reduces the risk of prediabetes and recurrence of MetS for up to 5 years.
Diabetic kidney disease (DKD) is an emerging pandemic, paralleling the worldwide increase in obesity and diabetes mellitus. DKD is now the most frequent cause of end-stage renal disease and is ...associated with an excessive risk of cardiovascular morbidity and mortality. DKD is a consequence of systemic endothelial dysfunction. The endothelial-dependent cytoprotective coagulation protease activated protein C (aPC) ameliorates glomerular damage in DKD, in part by reducing mitochondrial ROS generation in glomerular cells. Whether aPC reduces mitochondrial ROS generation in the tubular compartment remains unknown. Here, we conducted expression profiling of kidneys in diabetic mice (wild-type and mice with increased plasma levels of aPC, APChigh mice). The top induced pathways were related to metabolism and in particular to oxidoreductase activity. In tubular cells, aPC maintained the expression of genes related to the electron transport chain, PGC1-α expression, and mitochondrial mass. These effects were associated with reduced mitochondrial ROS generation. Likewise, NLRP3 inflammasome activation and sterile inflammation, which are known to be linked to excess ROS generation in DKD, were reduced in diabetic APChigh mice. Thus, aPC reduces mitochondrial ROS generation in tubular cells and dampens the associated renal sterile inflammation. These studies support approaches harnessing the cytoprotective effects of aPC in DKD.
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