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What Food Has the Highest Calcium?Calcium and inflammation -
Cell death was partially dependent on the presence of NLRP3 Fig. It has been suggested, that cell death—independent of IL-1β maturation and release—contributes to pathological damage in MSU-induced gouty arthritis The results indicate, that inhibition of CaSR-mediated CPP uptake or of lysosomal CPP breakdown lead to a significant inhibition of cell death Fig.
In addition, CaSR expression was found to be upregulated in the synovial lining layer in RA patients compared to osteoarthritis samples Fig. The result shown in Fig.
c CaSR protein expression was analyzed in serial sections of synovial membranes using anti-CaSR antibody red color. Sections were counterstained with hematoxylin.
Shown is one representative experiment out of six RA and two osteoarthritis, OA. f Calcium red staining with GBHA of cryostat sections of paws from collagen-induced arthritis CIA mice, black frame shows region of erosion zone.
Shown is one representative experiment out of six with three different mice. a , d , e , g Two-tailed Mann—Whitney U test a , d , e , g or Pearson correlation coefficient h , i was used.
Calcium Red staining of cryostat sections of paws showed positive staining at sites of bone erosions and on cartilage-deprived articular surfaces Fig. This is confirmed by our findings, that low fetuin-A concentrations are associated with exacerbated pro-inflammatory responses in vitro, while higher fetuin-A concentrations decrease the IL-1β production induced by excess CPP load.
The systemic clearance of CPPs and of excess minerals is realized by the reticuloendothelial system, specifically through liver sinusoidal endothelial cells for primary CPPs and via scavenger receptor A-expressing macrophages in liver and spleen for secondary CPPs In contrast, macropinocytotic uptake of CPPs by monocytes might be part of immune surveillance by the innate immune system as suggested recently MSU crystals and other crystalline salts are known to activate the NLRP3 inflammasome due to disruption of lysosomes We show here, that uptake of colloidal calciprotein nanoparticles does not induce lysosomal leakage.
Systemic hypercalcemia has also been reported to be associated with RA and linked to high disease activity 48 , although this is not generally believed to be a key feature of RA.
Anecdotal evidence indicates, that RA can lead to pronounced hypercalcemia if larger joints are involved In RA, bones of fingers and toes are often affected by periarticular demineralization, which occurs early in the disease before the erosive bone lesions develop.
Demineralization predicts later joint damage, is associated with inflammation, and can be prevented by efficient therapy with tumor necrosis factor TNF inhibitors 50 , 51 , 52 , At sites of osteoclastic bone resorption, fetuin-A is also available due to its abundance in mineralized bone 31 , and resorption of bone matrix is linked to release of [P i ] ex as well The resulting IL-1β release is also a pivotal factor for bone erosion in RA, since IL-1β knock-out completely protects TNF transgenic mice from the severe erosive disease typically present in this model In addition, IL-1β positive, pro-inflammatory monocytes have recently been confirmed to represent a cell state expanded in RA Instead, it is feasible that monocyte precursor cells in the bone marrow in RA are already exposed to increased calcium concentrations in the vicinity of arthritic joints, leading to increased receptor expression.
This mechanism might even contribute to the development of coronary heart disease CHD in general, since citrullinated proteins and PAD-4 enzyme can also be detected within atherosclerotic plaques obtained from non-RA patients 58 , while ACPA positivity also predicts the CHD risk in individuals not suffering from RA The pro-inflammatory effect mediated by CaSR stimulation and subsequent CPP-driven inflammation might explain the negative outcome of the EVOLVE trial 60 , in which CKD patients were treated with Cinacalcet.
Overload of this system is associated with pro-inflammatory responses from monocytes and macrophages including release of exorbitant IL-1β amounts, and could potentially be triggered by bone erosion, increased cell death or phosphate-rich diet.
In RA, the resulting monocyte activation followed by IL-1β release are detrimental due to the ensuing joint destruction. Age and sex matched healthy donors were recruited among healthy blood donors.
Monocytes were isolated from 75 RA patients to perform various experiments. The study was approved by the Ethics Review Board of the Medical Faculty, Leipzig University , , with written informed consent from all blood donors.
Peripheral blood mononuclear cells PBMCs were freshly isolated from human peripheral blood by density gradient centrifugation using Ficoll paque GE healthcare. Monocytes were directly stimulated after isolation in either standard RPMI cell culture medium high [P i ], 5.
Assays were performed in well plates. The following reagents and inhibitors were used for several cell culture experiments.
Na 2 HPO 4 , BaCl 2 , fetuin-A from FBS were purchased from Merck, CaCl 2 , sodium phosphonoformate tribasic hexahydrate, from Sigma, YM from Wako chemicals, Calhex, NPS, Latrunculin A, Cytochalasin D from Tocris, Latrunculin B, PAF C, LLOMe from CaymanChemicals, MgSO 4 from AppliChem, ATP from Roche, DMSO from Serva, N-fMLP from abcam, and CAMe from Selleck-Chem.
THP-1 cells were transduced with lentiviral Cas9 particles Dharmacon, Edit-R Lentiviral hEF1α-Blast-Cas9 Nuclease Particles, Cat.
The cells were selected for 1 week before further use. Blasticidin and puromycin Invitrogen concentrations for selection were established in advance by an antibiotic kill-curve. Cas9 protein expression was confirmed by Western Blot Cell Signaling, Cas9 antibody 7AA3, Cat.
VSGH or a non-targeting control sequence Edit-R Lentiviral mCMV-Puro non-targeting sgRNA particles, Cat. Further cell culture was the same as for THP-1 wildtype cells except for the addition of the two selection antibiotics.
To ensure that the transduced cells were not shedding virus particles, a p24 ELISA Sino Biologicals, Cat. Cells were plated as single cell clones in a well U-bottom plate on a BD FACS Aria III cell sorter Core Unit fluorescence technology, Leipzig University with the help of Kathrin Jäger.
After 3 weeks clones were screened in a Mismatch-Detection Assay Takara, Guide-it Mutation Detection Kit, Cat. One clone positive in the mismatch assay at the CASR site CaSR70 B6 and one non-targeting sgRNA clone not containing a mismatch Ctrl B6 were then used for further experiments.
See supplementary Fig. Deletion of base pairs at the target sequence was verified by Nextera DNA library preparation Illumina and sequencing of PCR products in the DNA core unit Leipzig University.
All sequences of CaSR70 B6 contained bp, bp, or bp deletions, which ruled out persistence of the wild-type gene, and were not present in Ctrl B6 cells see Supplementary Fig.
Mice were used at 2—8 months of age. The clinical severity of arthritis was quantified as follows: 0: no joint swelling, 1: swelling of one finger joint, 2: at least two swollen finger joints, 3: mild swelling of wrist or ankle, and 4: severe swelling of wrist and ankle.
Scores of all forepaws and hind paws were totaled for each mouse. Blood sampling by heart puncture, peritoneal lavage, bone marrow flushes from the cavities of femur bones, and clinical scoring of arthritis severity were all performed on day 40 post injection, when the mice were sacrificed. Arthritis severity was evaluated daily.
IL-1β in cell culture supernatants was measured with the mouse IL-1β ELISA BD Bioscience. To visualize inflammasome activation, ASC-speck formation was detected by immunological staining of ASC.
After washing with PBS, cells were imaged with a fluorescence microscope Zeiss Axio Observer. DMR was analyzed with the Corning ® EPIC ® Biosensor system and Epic Quest R 2. Analysis of monocytes was directly done after their isolation from peripheral blood. The same protocol was performed for THP-1 cells, which were seeded for differentiation at a density of 2.
The 2x CPP concentration was used for stimulation, if not indicated otherwise. Fetuin-A was detected with the goat-anti-Fetuin-A antibody N, Santa Cruz. CPP medium was filtrated with a Whatman ® Anotop ® sterile syringe filter 0. Mean main peak intensity or Z -average was used for approximation of particle size.
Data were analyzed with Malvern Zetasizer Software 7. Measurements were supported by the group of Stephanie Hoeppener, Jena Center for Soft Matter JCSM and the group of Lukas Wick Birgit Würz , Helmholtz Center for Environmental Research—UFZ Leipzig.
Most frequently images were recorded on an Olympus Soft Imaging Solution OSIS Megaview 1k or an Eagle 4k HS CCD camera system. Imaging processing was performed utilizing ImageJ 1. Particle characterization was performed utilizing cryo-TEM or utilizing sample blotting on carbon-coated TEM grids Quantifoil, Germany , respectively.
For these studies, 8. Samples were vitrified utilizing liquid ethane. After blotting and plunge freezing samples were transferred into a Gatan cryo-stage and maintained at liquid Nitrogen temperature until transferred to the TEM utilizing a Gatan cryo-holder.
Fifteen microliters of the solutions were blotted onto carbon-coated TEM grids and analyzed by TEM investigation. Initially a comparative study was performed to check if drying the particles on the TEM-grid does alter their shape or size. This was not the case and, hence, all studies on the particle size evolution, supplementing the DLS studies, were performed utilizing this approach.
Particle size analysis was performed by Origin 9. EDX spectra were acquired with a Bruker Quantax system. Qualitative mapping results are presented as a color-coded overlay of preselected elements. A low background FEI EDX holder was used to minimize background signals.
Naturally, the Cu originating from the utilized TEM grids and minor signals from the chamber could not be avoided. Selection of the elements was based on an overview scan with integrated element analysis in spectral mode.
Attempts to access the quantitative composition of the particles showed different results and were not further quantified in the present study. Spectra obtained on the individual particles were acquired by integrative scanning of the beam in STEM mode over the respective particle and integration of the obtained signals.
Finally, resin and DMP were mixed to obtain a curable resin. Slices were floated onto carbon-coated TEM grids Quantifoil. Post-staining was occasionally used to improve the contrast but was avoided in cases where EDX analysis was carried out to minimize the presence of signals originating from the stains.
Imaging of the slices was performed either in bright field-mode or by utilizing STEM to improve the contrast.
Particles have been characterized by cryo TEM Fig. EDX investigations were performed on two arbitrary selected areas containing several CPP nanoparticles, which were blotted after long incubation times of several days example shown in Fig.
Three independently prepared samples were investigated to study the CPP uptake in human monocytes. Uptake was confirmed in two of the samples stimulated with CPPs. At least two sample areas showing the uptake events were selected and further investigated by EDX mapping Fig.
Additionally, five to six individual CPPs were further analyzed by acquiring the full EDX spectra spectra for locations 1 and 4 are depicted in Fig. Locations of the analyzed CPPs are marked in the STEM image depicted in Fig. Confocal Raman imaging was used for the detection of calcium-phosphate nanoparticle uptake in monocytes.
Raman imaging was done with the help of Tom Venus in collaboration with the Institute of Medical Physics and Biophysics, Leipzig University.
Monocytes were imaged before and after adding 2. Images were taken and processed with the WiTec software Control FOUR and Project FOUR PLUS software. Addition of 0, 0. Cells were removed by centrifugation. Blots were washed with TBST thrice and developed with the ECL system. The intensity of the bands was measured using ImageJ 1.
Following incubation with secondary goat-anti-rabbit Ab conjugated with either peroxidase or phosphatase, stains were developed with the substrates 3,3-diaminobenzidine tetrahydrochloride DAB or 3-aminoethylcarbazole AEC , respectively.
Calcein-stained cell culture medium or calcein-stained CPPs were used to detect macropinocytosis of monocytes. Freshly isolated monocytes were seeded on 1. Calcein uptake was detected by flow cytometry LSR II, BD Biosciences, BD FACS Diva 8.
Data were analyzed with FlowJo V For analysis in WT and CaSR-deficient THP-1, cells were seeded on 1. THP-1 cells were washed with PBS and CPP uptake was detected by Amnis ® ImageStreamXMark II analysis. Data were analyzed with IDEAS 6. Magic Red Cathepsin-B Assay ImmunoChemistry Technologies was used for the detection of Cathepsin B activity in monocytes.
Lysosomal leakage was detected as described previously In brief, monocytes were seeded on 1. After washing, monocytes were resuspended in fresh cell culture medium and seeded in new agarose-coated plates.
Analysis was done with IDEAS 6. Pierce LDH Cytotoxicity Assay Kit Thermo Scientific was used to analyze cell death in THP-1 CaSR deficient and NLRP3-deficient cells. Maximal LDH release was detected by lysing cells before LDH measurement. Values of each experiment are represented as symbols in bars or in box-whisker-plots.
All graphs and statistics were prepared with GraphPad Prism 8. If not indicated otherwise asterisks indicate the statistical comparison to control conditions.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
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Specifically, they found that loss of Orai1 mitigated pain hypersensitivity in male mice, but not in female mice. This prompted the investigators to further investigate the causes of the sexual dimorphism of pain perception.
Using spinal cord electrophysiology techniques, they found that in male microglial Orai1 knockout mice, the potentiation, or an increase in strength, of synaptic transmission that occurs following nerve injury was reduced.
Male mice also showed reduced induction of inflammatory cytokines in spinal cord tissue in response to nerve injury, however this was not observed in female mice. Using a microglial marking technique called IBA1 staining, the investigators found that although microglial activation was increased in both male and female mice equally, inhibited Orai1 function reduced the extent of microglial activation in male but not female mice.
They then gave both male and female mice a small-molecule Orai1 inhibitor compound called CM, which is currently being tested in clinical trials, and found that while the drug mitigated neuropathic pain in male mice, it had no effect in the female mice. The findings demonstrate that Orai1 channels are key mediators of microglia-induced neuroinflammation and the sexually dimorphic role of microglia in neuropathic pain, underscoring the importance of developing sex-specific targeted therapies in the future.
Shogo Tsujikawa, PhD, a former post-doctoral fellow in the Prakriya laboratory, was co-first author of the study. Co-authors of the study include Apkar Apkarian, PhD , director of the Center for Translational Pain Research and professor of Neuroscience , of Anesthesiology and of Physical Medicine and Rehabilitation , and Megumi Yamashita, PhD, DDS , research assistant professor of Pharmacology.
This work was supported by National Institutes of Health grants R01NS, R21NS, P50 DA, and CIHR foundation grant FDN Type above and press Enter to search. Press Esc to cancel. Northwestern Medicine Northwestern University Faculty Profiles. News Center. Home » Calcium Channels Regulate Neuroinflammation and Neuropathic Pain.
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Calcium and inflammation arthritis and Strategies for digestive wellness risk factors increases the risk inflammation developing osteoporosis. Get the facts on infkammation right amounts of anx you wnd to protect bone Metabolism boosting drinks. The chronic Caldium of autoimmune Calcium and inflammation such as rheumatoid arthritis and Preventing stomach ulcers arthritis, as well as some drugs used to treat the conditions, raise osteoporosis risks. Women lose bone mineral density faster than men until age 65, when both sexes begin to lose bone at about the same rate. For women 19 to 50 years old the RDA is mg; those older than 50 should get 1, mg a day. Men should aim for 1, mg a day until they are 70, and afterwards increase their intake to 1, mg daily. Harmony the Capcium of our knowledge, no Strategies for digestive wellness has examined the effects of Cxlcium D-calcium cosupplementation Strategies for digestive wellness inflammatory biomarkers Strategies for digestive wellness adipocytokines in vitamin D-insufficient type 2 diabetics. This inflammatlon was performed to assess the effects of vitamin D and calcium supplementation on inflammatory biomarkers and adipocytokines in vitamin D-insufficient people with type 2 diabetes. Totally, diabetic patients were enrolled in this randomized, placebo-controlled clinical trial. Blood sampling was done for the quantification of inflammatory biomarkers and adipocytokines at the study baseline and after 8 weeks of intervention. Joint calcium-vitamin D supplementation might improve systemic inflammation through decreasing IL-6 and TNF-α concentrations in vitamin D-insufficient people with type 2 diabetes.
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