scholarly journals Pitting of malaria parasites in microfluidic devices mimicking spleen interendothelial slits

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aleix Elizalde-Torrent ◽  
Claudia Trejo-Soto ◽  
Lourdes Méndez-Mora ◽  
Marc Nicolau ◽  
Oihane Ezama ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Microfluidic Devices ◽  
Cell Integrity ◽  
Dapi Staining ◽  
Malaria Parasites ◽  
Hematopoietic Organ ◽  
Mechanical Constraints ◽  
Infected Rbcs ◽  
Quality Control Mechanism

AbstractThe spleen is a hematopoietic organ that participates in cellular and humoral immunity. It also serves as a quality control mechanism for removing senescent and/or poorly deformable red blood cells (RBCs) from circulation. Pitting is a specialized process by which the spleen extracts particles, including malaria parasites, from within circulating RBCs during their passage through the interendothelial slits (IES) in the splenic cords. To study this physiological function in vitro, we have developed two microfluidic devices modeling the IES, according to the hypothesis that at a certain range of mechanical stress on the RBC, regulated through both slit size and blood flow, would force it undergo the pitting process without affecting the cell integrity. To prove its functionality in replicating pitting of malaria parasites, we have performed a characterization of P. falciparum-infected RBCs (P.f.-RBCs) after their passage through the devices, determining hemolysis and the proportion of once-infected RBCs (O-iRBCs), defined by the presence of a parasite antigen and absence of DAPI staining of parasite DNA using a flow cytometry-based approach. The passage of P.f.-RBCs through the devices at the physiological flow rate did not affect cell integrity and resulted in an increase of the frequency of O-iRBCs. Both microfluidic device models were capable to replicate the pitting of P.f.-RBCs ex vivo by means of mechanical constraints without cellular involvement, shedding new insights on the role of the spleen in the pathophysiology of malaria.

scholarly journals Ex VivoMaturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites

2016 ◽  
Vol 60 (11) ◽  
pp. 6859-6866 ◽  
Author(s):  
Zi Wei Chang ◽  
Benoit Malleret ◽  
Bruce Russell ◽  
Laurent Rénia ◽  
Carla Claser
Keyword(s):  
Ex Vivo ◽  
Single Step ◽  
Parasite Development ◽  
Ring Stage ◽  
Malaria Parasites ◽  
Rodent Malaria ◽  
Content Type ◽  
Parasite Biology

ABSTRACTEx vivoassay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-termin vitroculture andex vivoantimalarial susceptibility assays are relatively cumbersome, relying onin vivopassage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stagePlasmodium berghei,P. yoelii, andP. vinckei vinckeiusing a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.

scholarly journals Magnetic Tracking of Protein Synthesis in Microfluidic Environments—Challenges and Perspectives

Nanomaterials ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 585 ◽  
Author(s):  
Wegener ◽  
Ennen ◽  
Walhorn ◽  
Anselmetti ◽  
Hütten ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cell Biology ◽  
Ex Vivo ◽  
Microfluidic Devices ◽  
Cellular Protein ◽  
Fine Tuning ◽  
Superparamagnetic Particles ◽  
Magnetic Tracking ◽  
Selection Of

A novel technique to study protein synthesis is proposed that uses magnetic nanoparticles in combination with microfluidic devices to achieve new insights into translational regulation. Cellular protein synthesis is an energy-demanding process which is tightly controlled and is dependent on environmental and developmental requirements. Processivity and regulation of protein synthesis as part of the posttranslational nano-machinery has now moved back into the focus of cell biology, since it became apparent that multiple mechanisms are in place for fine-tuning of translation and conditional selection of transcripts. Recent methodological developments, such as ribosome foot printing, propel current research. Here we propose a strategy to open up a new field of labelling, separation, and analysis of specific polysomes using superparamagnetic particles following pharmacological arrest of translation during cell lysis and subsequent analysis. Translation occurs in polysomes, which are assemblies of specific transcripts, associated ribosomes, nascent polypeptides, and other factors. This supramolecular structure allows for unique approaches to selection of polysomes by targeting the specific transcript, ribosomes, or nascent polypeptides. Once labeled with functionalized superparamagnetic particles, such assemblies can be separated in microfluidic devices or magnetic ratchets and quantified. Insights into the dynamics of translation is obtained through quantifying large numbers of ribosomes along different locations of the polysome. Thus, an entire new concept for in vitro, ex vivo, and eventually single cell analysis will be realized and will allow for magnetic tracking of protein synthesis.

scholarly journals Gallic acid affects intestinal-epithelial-cell integrity and selected amino-acid uptake in porcine in vitro and ex vivo permeability models

2020 ◽  
pp. 1-9
Author(s):  
Marco Tretola ◽  
Giuseppe Bee ◽  
Paolo Silacci
Keyword(s):  
Amino Acid ◽  
Epithelial Cell ◽  
Protein Expression ◽  
Gallic Acid ◽  
Intestinal Epithelial Cell ◽  
Ex Vivo ◽  
Amino Acid Transporter ◽  
Intestinal Epithelial ◽  
Cell Integrity

Abstract Gallic acid (GA) is widely used as a dietary supplement due to several health-promoting effects, although its effects on intestinal-epithelial-cell integrity and transport remain mostly unknown. The present study aims to clarify the effects of GA on tight junctions and intestinal nutrient uptake through in vitro and ex vivo models. Both intestinal porcine enterocyte cell line-J2 cells and porcine middle-jejunum segments were treated with 5 (T5), 25 (T25) and 50 (T50) µm GA and mounted in Ussing chambers to determine transepithelial resistance (TEER), claudin-1 (CLDN1), occludin (OCLN), zonula occludens-1 (ZO-1) protein (in tissues and cells) and mRNA (in cells) expression. In addition, uptake of l-glutamate (l-Glut), l-arginine (l-Arg), l-lysine (l-Lys) and l-methionine (l-Meth) together with cationic-amino-acid transporter-1 (CAT-1) and excitatory-amino-acid transporter-3 (EAAT3) expression was evaluated. No apoptosis was observed in GA-treated cells, but TEER and CLDN1 protein abundance was lower with T50 compared with untreated cells. l-Arg and l-Lys uptake was greater with T5 than with T25 and T50. Ex vivo, T50 decreased the TEER values and the protein levels of CLDN1, OCLN and ZO-1, whereas T5 and T25 only decreased CLDN1 protein expression compared with untreated tissues. Moreover, T25 increased l-Glut and l-Arg uptake, the latter confirmed by an increased protein expression of CAT-1. GA influences intestinal uptake of the tested cationic amino acids at low concentrations and decreases the intestinal-cell barrier function at high concentrations. Similarities were observed between in vitro and ex vivo, but different treatment times and structures must be considered.

scholarly journals Maturation of hematopoietic stem cells from prehematopoietic stem cells is accompanied by up-regulation of PD-L1

2017 ◽  
Vol 215 (2) ◽  
pp. 645-659 ◽  
Author(s):  
Joanna Tober ◽  
Marijke M.W. Maijenburg ◽  
Yan Li ◽  
Long Gao ◽  
Brandon K. Hadland ◽  
...  
Keyword(s):  
Stem Cells ◽  
Immune Responses ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Hematopoietic Organ ◽  
Programmed Death

Hematopoietic stem cells (HSCs) mature from pre-HSCs that originate in the major arteries of the embryo. To identify HSCs from in vitro sources, it will be necessary to refine markers of HSCs matured ex vivo. We purified and compared the transcriptomes of pre-HSCs, HSCs matured ex vivo, and fetal liver HSCs. We found that HSC maturation in vivo or ex vivo is accompanied by the down-regulation of genes involved in embryonic development and vasculogenesis, and up-regulation of genes involved in hematopoietic organ development, lymphoid development, and immune responses. Ex vivo matured HSCs more closely resemble fetal liver HSCs than pre-HSCs, but are not their molecular equivalents. We show that ex vivo–matured and fetal liver HSCs express programmed death ligand 1 (PD-L1). PD-L1 does not mark all pre-HSCs, but cell surface PD-L1 was present on HSCs matured ex vivo. PD-L1 signaling is not required for engraftment of embryonic HSCs. Hence, up-regulation of PD-L1 is a correlate of, but not a requirement for, HSC maturation.

scholarly journals Microfluidic Systems to Study Neutrophil Forward and Reverse Migration

2021 ◽  
Vol 12 ◽  
Author(s):  
Kehinde Adebayo Babatunde ◽  
Jose M. Ayuso ◽  
Sheena C. Kerr ◽  
Anna Huttenlocher ◽  
David J. Beebe
Keyword(s):  
Immune Cells ◽  
Ex Vivo ◽  
Microfluidic Devices ◽  
Resolution Of Inflammation ◽  
Effector Cells ◽  
Reverse Migration ◽  
Inflammatory Site ◽  
And Behavior

During infection, neutrophils are the most abundantly recruited innate immune cells at sites of infection, playing critical roles in the elimination of local infection and healing of the injury. Neutrophils are considered to be short-lived effector cells that undergo cell death at infection sites and in damaged tissues. However, recent in vitro and in vivo evidence suggests that neutrophil behavior is more complex and that they can migrate away from the inflammatory site back into the vasculature following the resolution of inflammation. Microfluidic devices have contributed to an improved understanding of the interaction and behavior of neutrophils ex vivo in 2D and 3D microenvironments. The role of reverse migration and its contribution to the resolution of inflammation remains unclear. In this review, we will provide a summary of the current applications of microfluidic devices to investigate neutrophil behavior and interactions with other immune cells with a focus on forward and reverse migration in neutrophils.

scholarly journals Novel Synthesized N-Ethyl-Piperazinyl-Amides of C2-Substituted Oleanonic and Ursonic Acids Exhibit Cytotoxic Effects through Apoptotic Cell Death Regulation

2021 ◽  
Vol 22 (20) ◽  
pp. 10967
Author(s):  
Oxana Kazakova ◽  
Alexandra Mioc ◽  
Irina Smirnova ◽  
Irina Baikova ◽  
Adrian Voicu ◽  
...  
Keyword(s):  
Cell Death ◽  
Ex Vivo ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Cancer Cell Line ◽  
Apoptotic Cell Death ◽  
Dapi Staining ◽  
Amide Derivatives ◽  
Induced Apoptosis

A series of novel hybrid chalcone N-ethyl-piperazinyl amide derivatives of oleanonic and ursonic acids were synthesized, and their cytotoxic potential was evaluated in vitro against the NCI-60 cancer cell line panel. Compounds 4 and 6 exhibited the highest overall anticancer activity, with GI50 values in some cases reaching nanomolar values. Thus, the two compounds were further assessed in detail in order to identify a possible apoptosis- and antiangiogenic-based mechanism of action induced by the assessed compounds. DAPI staining revealed that both compounds induced nuclei condensation and overall cell morphological changes consistent with apoptotic cell death. rtPCR analysis showed that up-regulation of pro-apoptotic Bak gene combined with the down-regulation of the pro-survival Bcl-XL and Bcl-2 genes caused altered ratios between the pro-apoptotic and anti-apoptotic proteins’ levels, leading to overall induced apoptosis. Molecular docking analysis revealed that both compounds exhibited high scores for Bcl-XL inhibition, suggesting that compounds may induce apoptotic cell death through targeted anti-apoptotic protein inhibition, as well. Ex vivo determinations showed that both compounds did not significantly alter the angiogenesis process on the tested cell lines.

scholarly journals Loss of the selective autophagy receptor p62 impairs murine myeloid leukemia progression and mitophagy

Blood ◽  
2019 ◽  
Vol 133 (2) ◽  
pp. 168-179 ◽  
Author(s):  
The Duy Nguyen ◽  
Shabnam Shaid ◽  
Olesya Vakhrusheva ◽  
Sebastian E. Koschade ◽  
Kevin Klann ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Cell Integrity ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Selective Autophagy ◽  
Mitochondrial Superoxide ◽  
Quality Control Mechanism

Abstract Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity.

Effects of the Tibetan herbal preparation PADMA 28 on blood lipids and lipid oxidisability in subjects with mild hypercholesterolaemia

VASA ◽  
2005 ◽  
Vol 34 (1) ◽  
pp. 11-17 ◽  
Author(s):  
Brunner-La Rocca ◽  
Schindler ◽  
Schlumpf ◽  
Saller ◽  
Suter
Keyword(s):  
Endothelial Dysfunction ◽  
Blood Lipids ◽  
Ex Vivo ◽  
Hdl Cholesterol ◽  
Blood Lipid ◽  
Tibetan Medicine ◽  
Double Blind ◽  
Intraarterial Infusion ◽  
Padma 28

Background: Previous studies showed an anti-atherosclerotic effect of PADMA 28, an herbal formula based on Tibetan medicine. As the mechanisms of action are not fully understood, we investigated whether PADMA 28 may lower blood lipids and lipid oxidisability, and affect early endothelial dysfunction. Patients and methods: Sixty otherwise healthy subjects with total cholesterol ≥5.2 mmol/l and < 8.0 mmol/l were randomly assigned to placebo or PADMA 28, 3 x 2 capsules daily, for 4 weeks (double-blind). Blood lipids (total, LDL-, and HDL-cholesterol, triglycerides, Apo-lipoprotein A1 and B) and ex vivo lipid oxidisability were measured before and after treatment. In a subset of 24 subjects, endothelial function was assessed using venous occlusion plethysmography with intraarterial infusion of acetylcholine. Isolated LDL and plasma both untreated and pre-treated with PADMA 28 extract were oxidised by the radical generator AAPH. Conjugated diene formation was measured at 245 nm. Results: Blood lipids did not change during the study in both groups. In contrast to previous reports in mild hypercholesterolaemia, no endothelial dysfunction was seen and, consequently, was not influenced by therapy. Ex vivo blood lipid oxidisability was significantly reduced with PADMA 28 (area under curve: 5.29 ± 1.62 to 4.99 ± 1.46, p = 0.01), and remained unchanged in the placebo group (5.33 ± 1.88 to 5.18 ± 1.78, p > 0.1). This effect persisted one week after cessation of medication. In vitro experiments confirmed the prevention of lipid peroxidation in the presence of PADMA 28 extracts. Persistent protection was also seen for LDL isolated from PADMA 28-pretreated blood after being subjected to rigorous purification. Conclusions: This study suggests that the inhibition of blood lipid oxidisability by PADMA 28 may play a role in its anti-atherosclerotic effect.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

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