Novel in vitro assays to detect circulating permeability factor(s) in idiopathic focal segmental glomerulosclerosis

2020 ◽  
Author(s):  
Dirk J W den Braanker ◽  
Rutger J Maas ◽  
Jeroen K Deegens ◽  
Cansu Yanginlar ◽  
Jack F M Wetzels ◽  
...  
Keyword(s):  
Focal Segmental Glomerulosclerosis ◽  
Calf Serum ◽  
In Vitro Assays ◽  
Future Research ◽  
Foetal Calf Serum ◽  
Dependent Manner ◽  
Segmental Glomerulosclerosis ◽  
Human And Mouse ◽  
Active Disease

Abstract Background Many patients with idiopathic focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation (TX). Although several circulating permeability factors (CPFs) responsible for recurrence have been suggested, there is no consensus. To facilitate CPF identification and predict recurrence after TX, there is a need for robust methods that demonstrate the presence of CPFs. Methods Cultured human podocytes (hPods) and human and mouse glomerular endothelial cells (ciGEnC, mGEnC) were exposed to plasmas of FSGS patients with presumed CPFs, and of (disease) controls. A visual scoring assay and flow cytometry analysis of side scatter were used to measured changes in cellular granularity after exposure to plasma. Results Nine out of 13 active disease plasmas of 10 FSGS patients with presumed CPFs induced granularity in hPod in a dose- and time-dependent manner. Corresponding remission plasmas induced no or less granularity in hPod. Similar results were obtained with ciGEnC and mGEnC, although induced granularity was less compared with hPod. Notably, foetal calf serum, healthy plasma and a remission plasma partially blocked FSGS plasma-induced hPod granularity. Conclusions We developed a novel assay in which active disease, presumably CPF-containing, FSGS plasmas induced granularity in cultured hPod. Our results may indicate the presence of CPF inhibitor(s) in healthy and remission plasma. We suggest the presence of a delicate balance between CPF and a CPF inhibitory factor, which is disturbed in patients with active disease. Our novel assays can be applied in future research to identify CPF and CPF inhibitors, and possibly to predict recurrence after TX.

scholarly journals Dose–Response Effects of 3-Nitrooxypropanol Combined with Low- and High-Concentrate Feed Proportions in the Dairy Cow Ration on Fermentation Parameters in a Rumen Simulation Technique

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1784
Author(s):  
Matthias Schilde ◽  
Dirk von Soosten ◽  
Liane Hüther ◽  
Susanne Kersten ◽  
Ulrich Meyer ◽  
...  
Keyword(s):  
Dose Response ◽  
Gas Production ◽  
Hydrogen Gas ◽  
Simulation Technique ◽  
Mitigation Strategies ◽  
Feed Additive ◽  
Future Research ◽  
Dependent Manner ◽  
Dose Dependent

Methane (CH4) from ruminal feed degradation is a major pollutant from ruminant livestock, which calls for mitigation strategies. The purpose of the present 4 × 2 factorial arrangement was to investigate the dose–response relationships between four doses of the CH4 inhibitor 3-nitrooxypropanol (3-NOP) and potential synergistic effects with low (LC) or high (HC) concentrate feed proportions (CFP) on CH4 reduction as both mitigation approaches differ in their mode of action (direct 3-NOP vs. indirect CFP effects). Diet substrates and 3-NOP were incubated in a rumen simulation technique to measure the concentration and production of volatile fatty acids (VFA), fermentation gases as well as substrate disappearance. Negative side effects on fermentation regarding total VFA and gas production as well as nutrient degradability were observed for neither CFP nor 3-NOP. CH4 production decreased from 10% up to 97% in a dose-dependent manner with increasing 3-NOP inclusion rate (dose: p < 0.001) but irrespective of CFP (CFP × dose: p = 0.094). Hydrogen gas accumulated correspondingly with increased 3-NOP dose (dose: p < 0.001). In vitro pH (p = 0.019) and redox potential (p = 0.066) varied by CFP, whereas the latter fluctuated with 3-NOP dose (p = 0.01). Acetate and iso-butyrate (mol %) decreased with 3-NOP dose, whereas iso-valerate increased (dose: p < 0.001). Propionate and valerate varied inconsistently due to 3-NOP supplementation. The feed additive 3-NOP was proven to be a dose-dependent yet effective CH4 inhibitor under conditions in vitro. The observed lack of additivity of increased CFP on the CH4 inhibition potential of 3-NOP needs to be verified in future research testing further diet types both in vitro and in vivo.

scholarly journals Role of leukemia inhibitor factor (LIF) in decidualisation of murine uterine stromal cells in vitro

2004 ◽  
Vol 181 (3) ◽  
pp. 477-492 ◽  
Author(s):  
AA Fouladi Nashta ◽  
CV Andreu ◽  
N Nijjar ◽  
JK Heath ◽  
SJ Kimber
Keyword(s):  
In Vitro Culture ◽  
Stromal Cells ◽  
Control Level ◽  
Calf Serum ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Alp Activity ◽  
Dose Dependent ◽  
In Cells

Decidualisation of uterine stromal cells is a prerequisite for implantation of the embryo in mice. Here we have used an in vitro culture system in which stromal cells decidualise as indicated by a number of markers, including an increase in alkaline phosphatase (ALP) activity. The latter was used as a quantitative marker of decidualisation in the presence of low (2%) fetal calf serum. Prostaglandin E(2) (PGE(2)), which is known to induce decidualisation, increased ALP activity, and this effect was blocked in a dose-dependent manner by indomethacin. Leukemia inhibitory factor (LIF) was then examined, but it had no effect on PGE(2) secretion. However, LIF suppressed ALP activity in a dose-dependent manner in the presence of 2% serum, while an inhibitor of LIF that competes for binding to its receptor reversed the effect of LIF and increased ALP activity above the control level. In serum-free cultures, stromal cells differentiated rapidly, and no differences were observed between LIF-treated and untreated cultures. Stromal cells produce LIF during in vitro culture, and this peaked at 48 h. Freshly collected stromal cells from both day-2 and -4 pregnant mice expressed mRNA for the LIF receptor, and the transcript level was higher in cells isolated on day 4. However, no differences were observed in the relative levels of transcripts in cells from day 2 and day 4 after culture, nor were there differences between the LIF-treated cultures and controls. Therefore, in this study, we have shown that LIF suppresses decidualisation of murine uterine stromal cells in the presence of serum, this is not due to the regulation of PGE(2) secretion by stromal cells.

scholarly journals Mechanical challenges and cytoskeletal impairments in focal segmental glomerulosclerosis

2018 ◽  
Vol 314 (5) ◽  
pp. F921-F925 ◽  
Author(s):  
Di Feng ◽  
Clark DuMontier ◽  
Martin R. Pollak
Keyword(s):  
Blood Flow ◽  
Focal Segmental Glomerulosclerosis ◽  
Kidney Injury ◽  
Future Research ◽  
Disease States ◽  
Filtration Barrier ◽  
Segmental Glomerulosclerosis ◽  
The Face ◽  
Foot Process ◽  
Future Research Directions

Focal segmental glomerulosclerosis (FSGS) is a histologically defined form of kidney injury typically mediated by podocyte dysfunction. Podocytes rely on their intricate actin-based cytoskeleton to maintain the glomerular filtration barrier in the face of mechanical challenges resulting from pulsatile blood flow and filtration of this blood flow. This review summarizes the mechanical challenges faced by podocytes in the form of stretch and shear stress, both of which may play a role in the progression of podocyte dysfunction and detachment. It also reviews how podocytes respond to these mechanical challenges in dynamic fashion through rearranging their cytoskeleton, triggering various biochemical pathways, and, in some disease states, altering their morphology in the form of foot process effacement. Furthermore, this review highlights the growing body of evidence identifying several mutations of important cytoskeleton proteins as causes of FSGS. Lastly, it synthesizes the above evidence to show that a better understanding of how these mutations leave podocytes vulnerable to the mechanical challenges they face is essential to better understanding the mechanisms by which they lead to disease. The review concludes with future research directions to fill this gap and some novel techniques with which to pursue these directions.

Drug activity against Onchocerca gutturosa males in vitro: a model for chemotherapeutic research on onchocerciasis

1987 ◽  
Vol 61 (4) ◽  
pp. 271-281 ◽  
Author(s):  
Simon Townson ◽  
C. Connelly ◽  
A. Dobinson ◽  
R. Muller
Keyword(s):  
Culture System ◽  
Serum Proteins ◽  
Good Condition ◽  
Calf Serum ◽  
New Drugs ◽  
Foetal Calf Serum ◽  
Partial Recovery ◽  
Feeder Cells ◽  
Compound Identification

ABSTRACTAn in vitro system for chemotherapeutic research using adult male Onchocerca gutturosa has been developed as a model for O. volvulus. Using a culture system consisting of medium MEM+10% heat inactivated foetal calf serum (IFCS)+LLCMK2 (monkey kidney) feeder cells in an atmosphere of 5% CO2 in air, we examined the effects of a range of antiparasitic drugs on worm motility. Ivermectin, levamisole, furapyrimidone, Mel W, chloroquine, metrifonate, flubendazole, amoscanate and the Ciba-Geigy compounds CGP 6140, CGP 20′376 and CGI 17658 either immobilized or significantly reduced motility levels at a concentration of 5x10−5M or less within a 7-day period. Worms were affected at very low concentrations by ivermectin (effective conc. to reduce motility levels to 50% of controls, 3.14x10−8M), levamisole (7.95x10−8M), CGP 6140 (8.87x10−9M) and CGP 20′376 (2.78x10−8M). Difficulties were experienced in accurately repeating the immotile endpoint for levamisole due to an inconsistent partial recovery of motility. Over a 7-day period diethylcarbamazine had little effect on motility levels, while suramin caused a slight increase in activity compared to controls at some timepoints. Subsequent experiments demonstrated some differences in drug efficacy depending on the presence or absence of serum and feeder cells in the culture system probably because of drug avidly binding to serum proteins. However, serum and cells were found to be essential ingredients of the culture system to maintain worms in good condition, indicating that new drugs should be evaluated both in the presence and absence of serum and cells. Comparisons were made between the responses of O. gutturosa and Brugia pahangi to certain drugs and these species were found to significantly differ in their sensitivities to ivermectin and a novel compound (Wellcome), indicating that Onchocerca parasites should be used wherever possible for compound identification and development intended for the treatment of onchocerciasis. The in vitro system described here, using male O. gutturosa, provides a basis for further research and a practical alternative to O. volvulus.

scholarly journals A Novel Kartogenin-Releasing Polymer Scaffold Promotes Wounded Rat Achilles Tendon Enthesis Healing

2018 ◽  
Vol 3 (3) ◽  
pp. 2473011418S0053
Author(s):  
Jianying Zhang ◽  
Daibang Nie ◽  
Guangyi Zhao ◽  
Susheng Tan ◽  
MaCalus Hogan ◽  
...  
Keyword(s):  
Achilles Tendon ◽  
Saline Group ◽  
Future Research ◽  
Dependent Manner ◽  
Polymer Scaffold ◽  
Normal Medium ◽  
Post Surgery ◽  
Tendon Stem Cells

Category: Hindfoot Introduction/Purpose: Entheses have a special fibrocartilage transition zone where tendons and ligaments attach to bone. Enthesis injury is very common, and the reattachment of tendon to bone is a great challenge because healing takes place between a soft tissue (tendon) and a hard tissue (bone). We have now developed a kartogene (KGN)-containing polymer scaffold (KGN-P) that can precisely deliver KGN to damaged enthesis area. The effects of the KGN-containing polymer on the healing of wounded TBJ were investigated in vitro and in vivo. Methods: The proliferation and chondrogenesis of rat Achilles tendon stem cells (TSCs) grown in four conditions were measured: normal medium (Control); normal medium with 100 nM KGN (KGN); lysine diisocyanate (LDI)-glycerol scaffold with normal medium (LDI-P); LDI-glycerol-KGN scaffold with normal medium (KGN-P).A wound (1 mm) was created on each hind leg Achilles enthesis of all 8 rats (3 months old). The wounds were then treated either with 10 ul saline (Wound); or 10 ul of 10 uM KGN (KGN); or LDI polymer scaffold (LDI-P); or KGN-containing polymer scaffold (KGN-P). The rats were sacrificed on day 15 and 30 post-surgery, and their Achilles entheses were collected for gross inspection and histochemical analysis. Results: KGN-containing polymers have sponge-like structures (Fig. 1A-D), and release KGN in a time- and temperature-dependent manner (Fig. 1E). KGN-P scaffold induced chondrogenesis of TSCs (Fig. 2D, 2H) without changing cell proliferation (Fig. 2I), and enhanced fibrocartilage-like tissue formation (Fig. 3E). KGN (Fig. 3C) and LDI-P (Fig. 3D) treated groups exhibited unhealed wound areas as in saline group (Fig. 3B). Finally, KGN-P and KGN treated rat TSCs underwent chondrogenesis by upregulating collagen II, aggrecan, and SOX-9 expression (Fig. 3F). Conclusion: Our results showed that KGN-containing polymer scaffold enhanced wounded enthesis healing by inducing TSC chondrogenesis and promoting the formation of the fibrocartilage in the wound site. The KGN-P may be used for regeneration of wounded entheses in clinical settings. Future research will focus on optimizing KGN concentration and releasing rate in the polymer scaffold during enthesis healing.

scholarly journals Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alan Sulpizio ◽  
Marena E Minelli ◽  
Min Wan ◽  
Paul D Burrowes ◽  
Xiaochun Wu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mass Spectrometry ◽  
Ubiquitin Ligase ◽  
Legionella Pneumophila ◽  
Kinase Activity ◽  
Effector Protein ◽  
Dependent Manner ◽  
Biochemical Analyses ◽  
Human And Mouse

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here, we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analyses demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

scholarly journals Kisspeptin-10 Inhibits Angiogenesis in Human Placental Vessels ex Vivo and Endothelial Cells in Vitro

Endocrinology ◽  
2010 ◽  
Vol 151 (12) ◽  
pp. 5927-5934 ◽  
Author(s):  
Thayalini Ramaesh ◽  
James J. Logie ◽  
Antonia K. Roseweir ◽  
Robert P. Millar ◽  
Brian R. Walker ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Vessels ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Biologically Active ◽  
Trophoblast Invasion ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Inhibition Of Proliferation

Recent studies suggest that kisspeptin (a neuropeptide central to the regulation of gonadotrophin secretion) has diverse roles in human physiology, including a putative role in implantation and placental function. Kisspeptin and its receptor are present in human blood vessels, where they mediate vasoconstriction, and kisspeptin is known to inhibit tumor metastasis and trophoblast invasion, both processes involving angiogenesis. We hypothesized that kisspeptin contributes to the regulation of angiogenesis in the reproductive system. The presence of the kisspeptin receptor was confirmed in human placental blood vessels and human umbilical vein endothelial cells (HUVEC) using immunochemistry. The ability of kisspeptin-10 (KP-10) (a shorter biologically active processed peptide) to inhibit angiogenesis was tested in explanted human placental arteries and HUVEC using complementary ex vivo and in vitro assays. KP-10 inhibited new vessel sprouting from placental arteries embedded in Matrigel and tube-like structure formation by HUVEC, in a concentration-dependent manner. KP-10 had no effect on HUVEC viability or apoptosis but induced concentration-dependent inhibition of proliferation and migration. In conclusion, KP-10 has antiangiogenic effects and, given its high expression in the placenta, may contribute to the regulation of angiogenesis in this tissue.

scholarly journals The Anti-Aging Potential of Neohesperidin and Its Synergistic Effects with Other Citrus Flavonoids in Extending Chronological Lifespan of Saccharomyces Cerevisiae BY4742

Molecules ◽  
2019 ◽  
Vol 24 (22) ◽  
pp. 4093 ◽  
Author(s):  
Chunxia Guo ◽  
Hua Zhang ◽  
Xin Guan ◽  
Zhiqin Zhou
Keyword(s):  
Synergistic Effects ◽  
Future Research ◽  
Dependent Manner ◽  
Ros Scavenging ◽  
Concentration Dependent Manner ◽  
Chronological Lifespan ◽  
Flavonoid Compounds ◽  
Citrus Flavonoids ◽  
High Throughput Assay

The anti-aging activity of many plant flavonoids, as well as their mechanisms of action, have been explored in the current literature. However, the studies on the synergistic effects between the different flavonoid compounds were quite limited in previous reports. In this study, by using a high throughput assay, we tested the synergistic effects between different citrus flavonoids throughout the yeast’s chronological lifespan (CLS). We studied the effect of four flavonoid compounds including naringin, hesperedin, hesperitin, neohesperidin, as well as their different combinations on the CLS of the yeast strain BY4742. Their ROS scavenging ability, in vitro antioxidant activity and the influence on the extracellular pH were also tested. The results showed that neohesperidin extended the yeast’s CLS in a concentration-dependent manner. Especially, we found that neohesperidin showed great potential in extending CLS of budding yeast individually or synergistically with hesperetin. The neohesperidin exhibited the strongest function in decreasing the reactive oxygen species (ROS) accumulation in yeast. These findings clearly indicated that neohesperidin is potentially an anti-aging citrus flavonoid, and its synergistic effect with other flavonoids on yeast’s CLS will be an interesting subject for future research of the anti-aging function of citrus fruits.

scholarly journals Anti-Inflammatory Effects of Formononetin 7-O-phosphate, a Novel Biorenovation Product, on LPS-Stimulated RAW 264.7 Macrophage Cells

Molecules ◽  
2019 ◽  
Vol 24 (21) ◽  
pp. 3910 ◽  
Author(s):  
Min-Seon Kim ◽  
Jin-Soo Park ◽  
You Chul Chung ◽  
Sungchan Jang ◽  
Chang-Gu Hyun ◽  
...  
Keyword(s):  
Biological Properties ◽  
In Vitro Assays ◽  
Raw 264.7 Cells ◽  
No Production ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Macrophage Cells ◽  
Anti Inflammatory ◽  
Reduced Toxicity

Biorenovation is a microbial enzyme-catalyzed structural modification of organic compounds with the potential benefits of reduced toxicity and improved biological properties relative to their precursor compounds. In this study, we synthesized a novel compound verified as formononetin 7-O-phosphate (FMP) from formononetin (FM) using microbial biotransformation. We further compared the anti-inflammatory properties of FMP to FM in lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells. We observed that cell viabilities and inhibitory effects on LPS-induced nitric oxide (NO) production were greater in FMP-treated RAW 264.7 cells than in their FM-treated counterparts. In addition, FMP treatment suppressed the production of proinflammatory cytokines such as prostaglandin-E2 (PGE2), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in a dose-dependent manner and concomitantly decreased the mRNA expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). We also found that FMP exerted its anti-inflammatory effects through the downregulation of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor kappa B (NF-κB) signaling pathways. In conclusion, we generated a novel anti-inflammatory compound using biorenovation and demonstrated its efficacy in cell-based in vitro assays.

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