scholarly journals Platelet generation from circulating megakaryocytes is triggered in the lung vasculature

2021 ◽  
Author(s):  
Xiaojuan Zhao ◽  
Dominic Alibhai ◽  
Tony Walsh ◽  
Nathalie Tarassova ◽  
Semra Birol ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Indirect Evidence ◽  
The Body ◽  
Lung Model ◽  
Pulmonary Vasculature ◽  
Mouse Heart ◽  
Main Site ◽  
Novel Approach ◽  
Large Numbers

Abstract Platelets are small anucleate blood cells1,2 with critical roles in haemostasis, thrombosis, inflammation, vascularization, innate immunity and tissue regeneration3,4. In vitro-derived platelets, as an alternative to native platelets, are attractive for fundamental research because of their rapid genetic tractability, as vectors for drug and genetic component delivery5 and in clinical platelet transfusion. At present, however, their very low production rate, and poor agonist responsiveness, are major obstacles. Platelets are formed by fragmentation from mature polyploid megakaryocytes (MKs), their precursor cells6, although the process of their generation remains incompletely understood7,8. Bone marrow is proposed to be the main site of platelet production, however indirect evidence since the 1930s9,10 and recent direct observation11 has shown that the lung can also be a primary site of platelet biogenesis. Here we established an ex vivo mouse heart-lung model (Fig. 1a) through which we were able to perfuse murine MKs. Remarkably, we could show for the first time that MKs, despite their large size, can pass multiple times through the lung vasculature, and that this leads to the generation of very large numbers of fully functional platelets (up to 3,000 per megakaryocyte7,12). Using this system and a novel in vitro microfluidic chamber we show roles for ventilation, oxygenation and healthy pulmonary endothelial cells in platelet generation. We show that MKs undergo enucleation upon repeated passage through pulmonary vasculature before fragmentation to generate platelets, with this final process dependent on the actin regulator TPM4. This advances our understanding of platelet formation in the body and establishes a novel approach to generate large numbers of them outside the body.

scholarly journals Platelet generation from circulating megakaryocytes is triggered in the lung vasculature

2021 ◽  
Author(s):  
Xiaojuan Zhao ◽  
Dominic Alibhai ◽  
Tony G. Walsh ◽  
Nathalie Tarassova ◽  
Semra Z. Birol ◽  
...  
Keyword(s):  
Blood Cells ◽  
Ex Vivo ◽  
Critical Role ◽  
The Body ◽  
Mouse Lung ◽  
Generation Process ◽  
Large Size ◽  
Large Numbers ◽  
Microfluidic Chamber

Platelets, small hemostatic blood cells, are derived from megakaryocytes, although the generation process is not clear. Only small numbers of platelets have been produced in systems outside the body, where bone marrow and lung are proposed as sites of platelet generation. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates very large numbers of platelets, up to 3,000 per megakaryocyte. Despite their large size, megakaryocytes were able repeatedly to passage through the lung vasculature, leading to enucleation and fragmentation to generate platelets intravascularly. Using the ex vivo lung and a novel in vitro microfluidic chamber we determined the contributions of oxygenation, ventilation and endothelial cell health to platelet generation, and showed a critical role for the actin regulator TPM4.

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.

scholarly journals Extracellular vesicles engineered with valency-controlled DNA nanostructures deliver CRISPR/Cas9 system for gene therapy

2020 ◽  
Vol 48 (16) ◽  
pp. 8870-8882 ◽  
Author(s):  
Jialang Zhuang ◽  
Jizhou Tan ◽  
Chenglin Wu ◽  
Jie Zhang ◽  
Ting Liu ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Primary Liver Cancer ◽  
The Body ◽  
Dna Aptamer ◽  
Great Promise ◽  
Tumor Growth Inhibition ◽  
Specific Cell ◽  
Dna Nanostructures

Abstract Extracellular vesicles (EVs) hold great promise for transporting CRISPR–Cas9 RNA-guided endonucleases (RNP) throughout the body. However, the cell-selective delivery of EVs is still a challenge. Here, we designed valency-controlled tetrahedral DNA nanostructures (TDNs) conjugated with DNA aptamer, and loaded the valency-controlled TDNs on EV surface via cholesterol anchoring for specific cell targeting. The targeting efficacy of different ratios of aptamer/cholesterol from 1:3 to 3:1 in TDNs on decorating EVs was investigated. TDNs with one aptamer and three cholesterol anchors (TDN1) efficiently facilitated the tumor-specific accumulation of the EVs in cultured HepG2 cells and human primary liver cancer-derived organoids, as well as xenograft tumor models. The intracellular delivery of RNP by TDN1-EVs successfully realized its subsequent genome editing, leading to the downregulation of GFP or WNT10B in specific cells. This system was ultimately applied to reduce the protein expression of WNT10B, which presented remarkable tumor growth inhibition in vitro, ex vivo and in vivo, and could be extended to other therapeutic targets. The present study provides a platform for the directional display of aptamer on surface labeling and the EVs-based Cas9 delivery, which provides a meaningful idea for future cell-selective gene editing.

scholarly journals ON THE NATURE OF BACTEREMIA IN EXPERIMENTAL PNEUMOCOCCAL PNEUMONIA IN THE DOG

1953 ◽  
Vol 97 (2) ◽  
pp. 297-314 ◽  
Author(s):  
Lucien A. Gregg ◽  
O. H. Robertson
Keyword(s):  
Pneumococcal Pneumonia ◽  
The Body ◽  
Bactericidal Action ◽  
Principal Mechanism ◽  
Shed Blood ◽  
Humoral Immune ◽  
Large Numbers ◽  
Experimental Canine

With the purpose of ascertaining the influence exerted by the pneumococcidal activity of the blood on the course of bacteremia occurring in experimental canine pneumococcal pneumonia, a study was made of the rates at which intravenously injected pneumococci disappeared from the circulation and the shed blood of diseased dogs. Preliminary studies on normal animals showed that blood containing hundreds of thousands of pneumococci per cc. immediately after injection usually became sterile or nearly so within an hour's time. Simultaneous observations carried out on the blood in vitro showed an analogous rapid disappearance of the microorganisms, although the effect was not quite as marked. Similar tests on non-bacteremic dogs with pneumonia revealed essentially the same ability of the body to dispose of large numbers of circulating pneumococci. The shed blood likewise exhibited marked bactericidal power. The occurrence of bacteremia during pneumonia did not retard greatly the rate at which injected pneumococci disappeared from the circulation, as compared with the non-bacteremic state. After several hours the numbers of circulating microorganisms were approximately the same as prior to the intravenous injection. Blood in vitro often cleared as fully as it did in vivo over the same length of time. Studies on the role played by humoral immune substances in the bactericidal action of the blood showed that while their presence was necessary for maximum killing power, and that bacteremic blood lacking humoral immune properties was rarely capable of self-sterilization in vitro, nevertheless such blood often retained considerable bactericidal potency as shown by its ability to reduce materially the numbers of pneumococci added to it. This phenomenon is discussed. The marked pneumococcidal capacity of the blood exhibited by dogs with experimental pneumococcal pneumonia and its persistence during bacteremia suggest that this constitutes the principal mechanism for limiting the degree of blood invasion. The similarity of the findings in canine and human pneumococcal lobar pneumonia is pointed out.

scholarly journals Anex vivocystic fibrosis model recapitulates key clinical aspects of chronicStaphylococcus aureusinfection

2019 ◽  
Author(s):  
Esther Sweeney ◽  
Marwa M. Hassan ◽  
Niamh E. Harrington ◽  
Alan R. Smyth ◽  
Matthew N. Hurley ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Ex Vivo ◽  
Lung Model ◽  
Clinical Aspects ◽  
Small Colony Variant ◽  
Tissue Invasion ◽  
Disease Characteristics ◽  
Small Colony ◽  
Clinically Significant

AbstractStaphylococcus aureusis one of the most prevalent organisms isolated from the airways of people with cystic fibrosis (CF), predominantly early in life. Yet its role in the pathology of lung disease is poorly understood. Clinical studies are limited in scope by age and health of participants andin vitrostudies are not always able to accurately recapitulate chronic disease characteristics such as the development of small colony variants. Further, animal models also do not fully represent features of clinical disease: in particular, mice are not readily colonized byS. aureusand when infection is established it leads to the formation of abscesses, a phenomenon almost never observed in the human CF lung. Here, we present details of the development of an existingex vivopig lung model of CF infection to investigate the growth ofS. aureus. We show thatS. aureusis able to establish infection and demonstrates clinically significant characteristics including small colony variant phenotype, increased antibiotic tolerance and preferential localisation in mucus. Tissue invasion and the formation of abscesses were not observed, in line with clinical data.

scholarly journals The continuing evolution of the Langendorff and ejecting murine heart: new advances in cardiac phenotyping

2012 ◽  
Vol 303 (2) ◽  
pp. H156-H167 ◽  
Author(s):  
Ronglih Liao ◽  
Bruno K. Podesser ◽  
Chee Chew Lim
Keyword(s):  
Vascular Reactivity ◽  
Ischemia Reperfusion Injury ◽  
Ex Vivo ◽  
Isolated Heart ◽  
Ischemia Reperfusion ◽  
In Vitro Assays ◽  
Mouse Heart ◽  
Heart Preparation ◽  
The Impact

The isolated retrograde-perfused Langendorff heart and the isolated ejecting heart have, over many decades, resulted in fundamental discoveries that form the underpinnings of our current understanding of the biology and physiology of the heart. These two experimental methodologies have proven invaluable in studying pharmacological effects on myocardial function, metabolism, and vascular reactivity and in the investigation of clinically relevant disease states such as ischemia-reperfusion injury, diabetes, obesity, and heart failure. With the advent of the genomics era, the isolated mouse heart preparation has gained prominence as an ex vivo research tool for investigators studying the impact of gene modification in the intact heart. This review summarizes the historical development of the isolated heart and provides a practical guide for the establishment of the Langendorff and ejecting heart preparations with a particular emphasis on the murine heart. In addition, current applications and novel methods of recording cardiovascular parameters in the isolated heart preparation will be discussed. With continued advances in methodological recordings, the isolated mouse heart preparation will remain physiologically relevant for the foreseeable future, serving as an integral bridge between in vitro assays and in vivo approaches.

scholarly journals Iodine-124 PET quantification of organ-specific delivery and expression of NIS-encoding RNA

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Matthias Miederer ◽  
Stefanie Pektor ◽  
Isabelle Miederer ◽  
Nicole Bausbacher ◽  
Isabell Sofia Keil ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ex Vivo ◽  
Small Animal ◽  
Protein Translation ◽  
The Body ◽  
Small Animal Pet ◽  
Animal Pet ◽  
Organ Specific

Abstract Background RNA-based vaccination strategies tailoring immune response to specific reactions have become an important pillar for a broad range of applications. Recently, the use of lipid-based nanoparticles opened the possibility to deliver RNA to specific sites within the body, overcoming the limitation of rapid degradation in the bloodstream. Here, we have investigated whether small animal PET/MRI can be employed to image the biodistribution of RNA-encoded protein. For this purpose, a reporter RNA coding for the sodium-iodide-symporter (NIS) was in vitro transcribed in cell lines and evaluated for expression. RNA-lipoplex nanoparticles were then assembled by complexing RNA with liposomes at different charge ratios, and functional NIS protein translation was imaged and quantified in vivo and ex vivo by Iodine-124 PET upon intravenous administration in mice. Results NIS expression was detected on the membrane of two cell lines as early as 6 h after transfection and gradually decreased over 48 h. In vivo and ex vivo PET/MRI of anionic spleen-targeting or cationic lung-targeting NIS-RNA lipoplexes revealed a visually detectable rapid increase of Iodine-124 uptake in the spleen or lung compared to control-RNA-lipoplexes, respectively, with minimal background in other organs except from thyroid, stomach and salivary gland. Conclusions The strong organ selectivity and high target-to-background acquisition of NIS-RNA lipoplexes indicate the feasibility of small animal PET/MRI to quantify organ-specific delivery of RNA.

scholarly journals The fate of methylmercury through the formation of bismethylmercury sulfide as an intermediate in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yumi Abiko ◽  
Yusuke Katayama ◽  
Wenyang Zhao ◽  
Sawako Horai ◽  
Kenji Sakurai ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Intraperitoneal Administration ◽  
Distal Colon ◽  
The Body ◽  
Gas Chromatography Mass Spectrometry ◽  
Dependent Manner ◽  
Free System ◽  
A Cell

AbstractA previous study by our group indicated that methylmercury (MeHg) is biotransformed to bismethylmercury sulfide [(MeHg)2S)] by interaction with reactive sulfur species (RSS) produced in the body. In the present study, we explored the transformation of MeHg to (MeHg)2S in the gut and the subsequent fate of (MeHg)2S in vitro and in vivo. An ex vivo experiment suggested the possibility of the extracellular transformation of MeHg to (MeHg)2S in the distal colon, and accordingly, the MeHg sulfur adduct was detected in the intestinal contents and feces of mice administered MeHg, suggesting that (MeHg)2S is formed through reactions between MeHg and RSS in the gut. In a cell-free system, we found that (MeHg)2S undergoes degradation in a time-dependent manner, resulting in the formation of mercury sulfide and dimethylmercury (DMeHg), as determined by X-ray diffraction and gas chromatography/mass spectrometry, respectively. We also identified DMeHg in the expiration after the intraperitoneal administration of (MeHg)2S to mice. Thus, our present study identified a new fate of MeHg through (MeHg)2S as an intermediate, which leads to conversion of volatile DMeHg in the body.

scholarly journals Ovarian Decellularized Bioscaffolds Provide an Optimal Microenvironment for Cell Growth and Differentiation In Vitro

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2126
Author(s):  
Georgia Pennarossa ◽  
Teresina De Iorio ◽  
Fulvio Gandolfi ◽  
Tiziana A. L. Brevini
Keyword(s):  
Ex Vivo ◽  
Fertility Restoration ◽  
Dermal Fibroblasts ◽  
Decellularized Extracellular Matrix ◽  
Ovarian Cells ◽  
Novel Approach ◽  
Definitive Solution ◽  
Cell Growth And Differentiation ◽  
Ex Vivo Culture

Ovarian failure is the most common cause of infertility. Although numerous strategies have been proposed, a definitive solution for recovering ovarian functions and restoring fertility is currently unavailable. One innovative alternative may be represented by the development of an “artificial ovary” that could be transplanted in patients for re-establishing reproductive activities. Here, we describe a novel approach for successful repopulation of decellularized ovarian bioscaffolds in vitro. Porcine whole ovaries were subjected to a decellularization protocol that removed the cell compartment, while maintaining the macrostructure and microstructure of the original tissue. The obtained bioscaffolds were then repopulated with porcine ovarian cells or with epigenetically erased porcine and human dermal fibroblasts. The results obtained demonstrated that the decellularized extracellular matrix (ECM)-based scaffold may constitute a suitable niche for ex vivo culture of ovarian cells. Furthermore, it was able to properly drive epigenetically erased cell differentiation, fate, and viability. Overall, the method described represents a powerful tool for the in vitro creation of a bioengineered ovary that may constitute a promising solution for hormone and fertility restoration. In addition, it allows for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies.

scholarly journals Repurposing ciclopirox as a pharmacological chaperone in a model of congenital erythropoietic porphyria

2018 ◽  
Vol 10 (459) ◽  
pp. eaat7467 ◽  
Author(s):  
Pedro Urquiza ◽  
Ana Laín ◽  
Arantza Sanz-Parra ◽  
Jorge Moreno ◽  
Ganeko Bernardo-Seisdedos ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Ex Vivo ◽  
The Body ◽  
Missense Mutations ◽  
Allosteric Site ◽  
Cutaneous Lesions ◽  
Pharmacological Chaperone ◽  
Genetic Mouse Model ◽  
Congenital Erythropoietic Porphyria

Congenital erythropoietic porphyria is a rare autosomal recessive disease produced by deficient activity of uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway. The disease affects many organs, can be life-threatening, and currently lacks curative treatments. Inherited mutations most commonly reduce the enzyme’s stability, altering its homeostasis and ultimately blunting intracellular heme production. This results in uroporphyrin by-product accumulation in the body, aggravating associated pathological symptoms such as skin photosensitivity and disfiguring phototoxic cutaneous lesions. We demonstrated that the synthetic marketed antifungal ciclopirox binds to the enzyme, stabilizing it. Ciclopirox targeted the enzyme at an allosteric site distant from the active center and did not affect the enzyme’s catalytic role. The drug restored enzymatic activity in vitro and ex vivo and was able to alleviate most clinical symptoms of congenital erythropoietic porphyria in a genetic mouse model of the disease at subtoxic concentrations. Our findings establish a possible line of therapeutic intervention against congenital erythropoietic porphyria, which is potentially applicable to most of deleterious missense mutations causing this devastating disease.

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