12 research outputs found
Classification of Microglial Morphological Phenotypes Using Machine Learning
Microglia are the brain’s immunocompetent macrophages with a unique feature that
allows surveillance of the surrounding microenvironment and subsequent reactions to
tissue damage, infection, or homeostatic perturbations. Thereby, microglia’s striking
morphological plasticity is one of their prominent characteristics and the categorization of
microglial cell function based on morphology is well established. Frequently, automated
classification of microglial morphological phenotypes is performed by using quantitative
parameters. As this process is typically limited to a few and especially manually chosen
criteria, a relevant selection bias may compromise the resulting classifications. In our
study, we describe a novel microglial classification method by morphological evaluation
using a convolutional neuronal network on the basis of manually selected cells in addition
to classical morphological parameters. We focused on four microglial morphologies,
ramified, rod-like, activated and amoeboid microglia within the murine hippocampus
and cortex. The developed method for the classification was confirmed in a mouse
model of ischemic stroke which is already known to result in microglial activation
within affected brain regions. In conclusion, our classification of microglial morphological
phenotypes using machine learning can serve as a time-saving and objective method
for post-mortem characterization of microglial changes in healthy and disease mouse
models, and might also represent a useful tool for human brain autopsy samples
Plant Sterol-Poor Diet Is Associated with Pro-Inflammatory Lipid Mediators in the Murine Brain
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 -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 -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
Classification of Microglial Morphological Phenotypes Using Machine Learning
Microglia are the brain’s immunocompetent macrophages with a unique feature that
allows surveillance of the surrounding microenvironment and subsequent reactions to
tissue damage, infection, or homeostatic perturbations. Thereby, microglia’s striking
morphological plasticity is one of their prominent characteristics and the categorization of
microglial cell function based on morphology is well established. Frequently, automated
classification of microglial morphological phenotypes is performed by using quantitative
parameters. As this process is typically limited to a few and especially manually chosen
criteria, a relevant selection bias may compromise the resulting classifications. In our
study, we describe a novel microglial classification method by morphological evaluation
using a convolutional neuronal network on the basis of manually selected cells in addition
to classical morphological parameters. We focused on four microglial morphologies,
ramified, rod-like, activated and amoeboid microglia within the murine hippocampus
and cortex. The developed method for the classification was confirmed in a mouse
model of ischemic stroke which is already known to result in microglial activation
within affected brain regions. In conclusion, our classification of microglial morphological
phenotypes using machine learning can serve as a time-saving and objective method
for post-mortem characterization of microglial changes in healthy and disease mouse
models, and might also represent a useful tool for human brain autopsy samples
Classification of Microglial Morphological Phenotypes Using Machine Learning
Microglia are the brain’s immunocompetent macrophages with a unique feature that
allows surveillance of the surrounding microenvironment and subsequent reactions to
tissue damage, infection, or homeostatic perturbations. Thereby, microglia’s striking
morphological plasticity is one of their prominent characteristics and the categorization of
microglial cell function based on morphology is well established. Frequently, automated
classification of microglial morphological phenotypes is performed by using quantitative
parameters. As this process is typically limited to a few and especially manually chosen
criteria, a relevant selection bias may compromise the resulting classifications. In our
study, we describe a novel microglial classification method by morphological evaluation
using a convolutional neuronal network on the basis of manually selected cells in addition
to classical morphological parameters. We focused on four microglial morphologies,
ramified, rod-like, activated and amoeboid microglia within the murine hippocampus
and cortex. The developed method for the classification was confirmed in a mouse
model of ischemic stroke which is already known to result in microglial activation
within affected brain regions. In conclusion, our classification of microglial morphological
phenotypes using machine learning can serve as a time-saving and objective method
for post-mortem characterization of microglial changes in healthy and disease mouse
models, and might also represent a useful tool for human brain autopsy samples
Plant Sterol-Poor Diet Is Associated with Pro-Inflammatory Lipid Mediators in the Murine Brain
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 -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 -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
Plant Sterol-Poor Diet Is Associated with Pro-Inflammatory Lipid Mediators in the Murine Brain
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 -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 -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
Plant Sterol-Poor Diet Is Associated with Pro-Inflammatory Lipid Mediators in the Murine Brain
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
Articular cartilage chondrocytes express aromatase and use enzymes involved in estrogen metabolism
Introduction
Sex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17β-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue.
Methods
We analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes.
Results
Aromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10−11 M to 10−7 M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-β. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole.
Conclusions
We demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-β. In addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated
Synergistic Highly Potent Targeted Drug Combinations in different Pheochromocytoma Models including Human Tumor Cultures
There are no officially approved therapies for metastatic pheochromocytomas apart from ultratrace 131I-MIBG therapy approved only in the United States. We have, therefore, investigated the anti-tumor potential of novel molecular-targeted approaches in murine pheochromocytoma cell lines (MPC/MTT), immortalized mouse chromaffin Sdhb-/- cells, 3D-pheochromocytoma tumor models (MPC/MTT spheroids) and human pheochromocytoma primary cultures. We identified the specific PI3Kα inhibitor BYL719 and the mTORC1 inhibitor everolimus as the most effective combination in all models. Single treatment with clinically relevant doses of BYL719 and everolimus significantly decreased MPC/MTT and Sdhb-/- cell viability. A targeted combination of both inhibitors synergistically reduced MPC and Sdhb-/- cell viability and showed an additive effect on MTT cells. In MPC/MTT spheroids, treatment with clinically relevant doses of BYL719 alone or in combination with everolimus was highly effective, leading to a significant shrinkage or even a complete collapse of the spheroids. We confirmed the synergism of clinically relevant doses of BYL719 plus everolimus in human pheochromocytoma primary cultures of individual patient tumors with BYL719 attenuating everolimus-induced AKT activation. We have thus established a method to assess molecular-targeted therapies in human pheochromocytoma cultures and identified a highly effective combination therapy. Our data pave the way to customized combination therapy to target individual patient tumors