scholarly journals Inhibition of Chondroitin-4-Sulfate-Specific Adhesion of Plasmodium falciparum-Infected Erythrocytes by Sulfated Polysaccharides

2005 ◽  
Vol 73 (7) ◽  
pp. 4288-4294 ◽  
Author(s):  
Katherine T. Andrews ◽  
Nicole Klatt ◽  
Yvonne Adams ◽  
Petra Mischnick ◽  
Reinhard Schwartz-Albiez
Keyword(s):  
Plasmodium Falciparum ◽  
Ex Vivo ◽  
Severe Disease ◽  
Placental Tissue ◽  
Peripheral Circulation ◽  
Sulfated Polysaccharides ◽  
Mature Stage ◽  
Dependent Manner ◽  
Inhibitory Compounds

ABSTRACT Adhesion of Plasmodium falciparum-infected erythrocytes to placental chondroitin 4-sulfate (CSA) has been linked to the severe disease outcome of pregnancy-associated malaria. Soluble polysaccharides that release mature-stage parasitized erythrocytes into the peripheral circulation may help elucidate these interactions and have the potential to aid in developing therapeutic strategies. We have screened a panel of 11 sulfated polysaccharides for their capacities to inhibit adhesion of infected erythrocytes to CSA expressed on CHO-K1 cells and ex vivo human placental tissue. Two carrageenans and a cellulose sulfate (CS10) were able to inhibit adhesion to CSA and to cause already bound infected erythrocytes to de-adhere in a dose-dependent manner. CS10, like CSA and in contrast to all other compounds tested, remained bound to infected erythrocytes after washing and continued to inhibit binding. Both carrageenans and CS10 inhibited adhesion to placental tissue. Although highly sulfated dextran sulfate can inhibit CSA-mediated adhesion to CHO cells, this polysaccharide amplified adhesion to placental tissue severalfold, demonstrating the importance of evaluating inhibitory compounds in systems as close to in vivo as possible. Interestingly, and in contrast to all other compounds tested, which had a random distribution of sulfate groups, CS10 exhibited a clustered sulfate pattern along the polymer chain, similar to that of the undersulfated placental CSA preferred by placental-tissue-binding infected erythrocytes. Therefore, the specific antiadhesive capacity observed here seems to depend not only on the degree of charge and sulfation but also on a particular pattern of sulfation.

Ethanol Extract of Achillea fragrantissima Enhances Angiogenesis through Stimulation of VEGF Production

2020 ◽  
Vol 21 ◽  
Author(s):  
Hana M. Hammad ◽  
Amer Imraish ◽  
Maysa Al-Hussaini ◽  
Malek Zihlif ◽  
Amani A. Harb ◽  
...  
Keyword(s):  
Wound Healing ◽  
Rural Communities ◽  
Ex Vivo ◽  
Ethanol Extract ◽  
Aortic Ring ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Achillea Fragrantissima

Objective: Achillea fragrantissima L. (Asteraceae) is a traditionally used medicinal herb in the rural communities of Jordan. Methods: The present study evaluated the efficacy of the ethanol extract of this species on angiogenesis in both, ex vivo using rat aortic ring assay and in vivo using rat excision wound model. Results: In concentrations of 50 and 100 µg/ml, the ethanol extract showed angiogenic stimulatory effect and significantly increased length of capillary protrusions around aorta rings of about 60% in comparison to those of untreated aorta rings. In MCF-7 cells, the ethanol extract of A. fragrantissima stimulates the production of VEGF in a dose-dependent manner. 1% and 5% of ethanol extract of A. fragrantissima containing vaseline based ointment was applied on rat excision wounds for six days and was found to be effective in wound healing and maturation of the scar. Both preparations resulted in better wound healing when compared to the untreated control group and vaseline-treated group. This effect was comparable to that induced by MEBO, the positive control. Conclusion: The results indicate that A. fragrantissima has a pro-angiogenic effect, which may act through the VEGF signaling pathway.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals Transcriptomic Analysis of Inbred Chicken Lines Reveals Infectious Bursal Disease Severity Is Associated with Greater Bursal Inflammation In Vivo and More Rapid Induction of Pro-Inflammatory Responses in Primary Bursal Cells Stimulated Ex Vivo

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 933
Author(s):  
Amin S. Asfor ◽  
Salik Nazki ◽  
Vishwanatha R.A.P. Reddy ◽  
Elle Campbell ◽  
Katherine L. Dulwich ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Severe Disease ◽  
Infectious Bursal Disease ◽  
Rna Seq ◽  
Rapid Induction ◽  
Cytoskeletal Regulation ◽  
Bursal Disease

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p < 0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

scholarly journals Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 730
Author(s):  
Biji Mathew ◽  
Leianne A. Torres ◽  
Lorea Gamboa Gamboa Acha ◽  
Sophie Tran ◽  
Alice Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Extracellular Vesicles ◽  
Time Course ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Paracrine Effects

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

scholarly journals Transcriptomic analysis reveals that severity of infectious bursal disease in White Leghorn inbred chicken lines is associated with greater bursal inflammation in vivo and more rapid induction of pro-inflammatory responses in primary bursal cells stimulated ex vivo.

2021 ◽  
Author(s):  
Amin S Asfor ◽  
Salik Nazki ◽  
Vishwanatha RAP Reddy ◽  
Elle Campbell ◽  
Katherine L Dulwich ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Severe Disease ◽  
Infectious Bursal Disease ◽  
White Leghorn ◽  
Rapid Induction ◽  
Cytoskeletal Regulation ◽  
Bursal Disease

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p<0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

Skeletal muscle phenotype signaling with ex vivo endurance-type dynamic contractions in rat muscle

2021 ◽  
Author(s):  
Jesper Emil Jakobsgaard ◽  
Jacob Andresen ◽  
Frank V. de Paoli ◽  
Kristian Vissing
Keyword(s):  
Skeletal Muscle ◽  
Translation Initiation ◽  
Ex Vivo ◽  
Contractile Activity ◽  
Specific Protein ◽  
Signaling Proteins ◽  
Dependent Manner ◽  
Metabolic Signaling ◽  
Dynamic Endurance

Skeletal muscle phenotype may influence the response sensitivity of myocellular regulatory mechanisms to contractile activity. To examine this, we employed an ex vivo endurance-type dynamic contraction model to evaluate skeletal muscle phenotype-specific protein signaling responses in rat skeletal muscle. Preparations of slow-twitch soleus and fast-twitch extensor digitorum longus skeletal muscle from 4-wk old female Wistar rats were exposed to an identical ex vivo dynamic endurance-type contraction paradigm consisting of 40 minutes of stretch-shortening contractions under simultaneous low-frequency electrostimulation delivered in an intermittent pattern. Phosphorylation of proteins involved in metabolic signaling and signaling for translation initiation was evaluated at 0, 1, and 4 hours after stimulation by immunoblotting. For both muscle phenotypes, signaling related to metabolic events was upregulated immediately after stimulation, with concomitant absence of signaling for translation-initiation. Signaling for translation-initiation was then activated in both muscle phenotypes at 1-4 hours after stimulation, coinciding with attenuated metabolic signaling. The recognizable pattern of signaling responses support how our ex vivo dynamic muscle contraction model can be utilized to infer a stretch-shortening contraction pattern resembling stretch-shortening contraction of in vivo endurance exercise. Moreover, using this model, we observed that some specific signaling proteins adhering to metabolic events or to translation initation exhibited phosphorylation changes in a phenotype-dependent manner, whereas other signaling proteins exhibited phenotype-independent changes. These findings may aid the interpretation of myocellular signaling outcomes adhering to mixed muscle samples collected during human experimental trials.

scholarly journals The NADPH Oxidase Nox4 Controls Macrophage Polarization in an NFκB-Dependent Manner

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
V. Helfinger ◽  
K. Palfi ◽  
A. Weigert ◽  
K. Schröder
Keyword(s):  
Ex Vivo ◽  
Macrophage Polarization ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Superoxide Anions ◽  
Wild Type ◽  
Macrophage Population ◽  
The Family ◽  
High Level

The family of NADPH oxidases represents an important source of reactive oxygen species (ROS) within the cell. Nox4 is a special member of this family as it constitutively produces H2O2 and its loss promotes inflammation. A major cellular component of inflammation is the macrophage population, which can be divided into several subpopulations depending on their phenotype, with proinflammatory M(LPS+IFNγ) and wound-healing M(IL4+IL13) macrophages being extremes of the functional spectrum. Whether Nox4 is expressed in macrophages is discussed controversially. Here, we show that macrophages besides a high level of Nox2 indeed express Nox4. As Nox4 contributes to differentiation of many cells, we hypothesize that Nox4 plays a role in determining the polarization and the phenotype of macrophages. In bone marrow-derived monocytes, ex vivo treatment with LPS/IFNγ or IL4/IL13 results in polarization of the cells into M(LPS+IFNγ) or M(IL4+IL13) macrophages, respectively. In this ex vivo setting, Nox4 deficiency reduces M(IL4+IL13) polarization and forces M(LPS+IFNγ). Nox4-/- M(LPS+IFNγ)-polarized macrophages express more Nox2 and produce more superoxide anions than wild type M(LPS+IFNγ)-polarized macrophages. Mechanistically, Nox4 deficiency reduces STAT6 activation and promotes NFκB activity, with the latter being responsible for the higher level of Nox2 in Nox4-deficient M(LPS+IFNγ)-polarized macrophages. According to those findings, in vivo, in a murine inflammation-driven fibrosarcoma model, Nox4 deficiency forces the expression of proinflammatory genes and cytokines, accompanied by an increase in the number of proinflammatory Ly6C+ macrophages in the tumors. Collectively, the data obtained in this study suggest an anti-inflammatory role for Nox4 in macrophages. Nox4 deficiency results in less M(IL4+IL13) polarization and suppression of NFκB activity in monocytes.

scholarly journals Peptidic degron for IMiD-induced degradation of heterologous proteins

2019 ◽  
Vol 116 (7) ◽  
pp. 2539-2544 ◽  
Author(s):  
Vidyasagar Koduri ◽  
Samuel K. McBrayer ◽  
Ella Liberzon ◽  
Adam C. Wang ◽  
Kimberly J. Briggs ◽  
...  
Keyword(s):  
Protein Function ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Ease Of Use ◽  
Dependent Manner ◽  
Heterologous Proteins ◽  
Ubiquitin Ligase Complex ◽  
Dose Dependent ◽  
Current Systems

Current systems for modulating the abundance of proteins of interest in living cells are powerful tools for studying protein function but differ in terms of their complexity and ease of use. Moreover, no one system is ideal for all applications, and the best system for a given protein of interest must often be determined empirically. The thalidomide-like molecules (collectively called the IMiDs) bind to the ubiquitously expressed cereblon ubiquitin ligase complex and alter its substrate specificity such that it targets the IKZF1 and IKZF3 lymphocyte transcription factors for destruction. Here, we mapped the minimal IMiD-responsive IKZF3 degron and show that this peptidic degron can be used to target heterologous proteins for destruction with IMiDs in a time- and dose-dependent manner in cultured cells grown ex vivo or in vivo.

scholarly journals Opposing effects of nitric oxide and prostaglandin inhibition on muscle mitochondrial V̇o2 during exercise

2012 ◽  
Vol 303 (1) ◽  
pp. R94-R100 ◽  
Author(s):  
Robert Boushel ◽  
Teresa Fuentes ◽  
Ylva Hellsten ◽  
Bengt Saltin
Keyword(s):  
Nitric Oxide ◽  
Blood Flow ◽  
Mitochondrial Respiration ◽  
Complex I ◽  
Ex Vivo ◽  
Knee Extension ◽  
Physiological Effect ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Nitric oxide (NO) and prostaglandins (PG) together play a role in regulating blood flow during exercise. NO also regulates mitochondrial oxygen consumption through competitive binding to cytochrome- c oxidase. Indomethacin uncouples and inhibits the electron transport chain in a concentration-dependent manner, and thus, inhibition of NO and PG synthesis may regulate both muscle oxygen delivery and utilization. The purpose of this study was to examine the independent and combined effects of NO and PG synthesis blockade (l-NMMA and indomethacin, respectively) on mitochondrial respiration in human muscle following knee extension exercise (KEE). Specifically, this study examined the physiological effect of NO, and the pharmacological effect of indomethacin, on muscle mitochondrial function. Consistent with their mechanism of action, we hypothesized that inhibition of nitric oxide synthase (NOS) and PG synthesis would have opposite effects on muscle mitochondrial respiration. Mitochondrial respiration was measured ex vivo by high-resolution respirometry in saponin-permeabilized fibers following 6 min KEE in control (CON; n = 8), arterial infusion of NG-monomethyl-l-arginine (l-NMMA; n = 4) and Indo ( n = 4) followed by combined inhibition of NOS and PG synthesis (l-NMMA + Indo, n = 8). ADP-stimulated state 3 respiration (OXPHOS) with substrates for complex I (glutamate, malate) was reduced 50% by Indo. State 3 O2 flux with complex I and II substrates was reduced less with both Indo (20%) and l-NMMA + Indo (15%) compared with CON. The results indicate that indomethacin reduces state 3 mitochondrial respiration primarily at complex I of the respiratory chain, while blockade of NOS by l-NMMA counteracts the inhibition by Indo. This effect on muscle mitochondria, in concert with a reduction of blood flow accounts for in vivo changes in muscle O2 consumption during combined blockade of NOS and PG synthesis.

Nicotinamide Enhances the Polyploidization of Primary Megakaryocytes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1366-1366
Author(s):  
Lisa M. Giammona ◽  
Eleftherios Papoutsakis ◽  
William M. Miller
Keyword(s):  
Dna Content ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Annexin V ◽  
Dependent Manner ◽  
Vitamin B3 ◽  
Ploidy Levels ◽  
High Ploidy

Abstract Megakaryocyte (Mk) maturation includes the development of polyploid cells via endomitosis. In vitro models of Mk differentiation can be used to gain a better understanding of the molecular mechanisms controlling this process. However, it is challenging to achieve ploidy levels in cultured human cells that are as high as those observed in vivo. Others have recently reported the use of chemical inhibitors to increase Mk ploidy (Lannutti et al., Blood 105:3875, 2005). Here, we show that nicotinamide (NIC), a form of vitamin B3, enhances the normal process of Mk polyploidization and leads to both a greater fraction of high ploidy cells and a greater degree of polyploidization. Human mobilized peripheral blood CD34+ cells were cultured in serum-free medium supplemented with thrombopoietin (TPO) to induce Mk differentiation. Beginning on day 5 of culture, cells were treated with nicotinamide (3 and 6.25 mM) and monitored for DNA content, growth, apoptosis, and surface marker expression. NIC treatment resulted in a greater fraction of Mks with high ploidy (DNA content greater than or equal to 8N). The ploidy of NIC treated cells continued to increase over the duration of the 13-day culture, whereas the ploidy of untreated cells peaked at day 9. On day 13 (8 days of NIC exposure), the percentages of high ploidy Mks for the untreated, 3 mM NIC, and 6.25 mM NIC conditions were 23%, 48%, and 63%, respectively. Furthermore, cells treated with NIC reached ploidy levels of 64N and 32N for 6.25 and 3 mM NIC, respectively, compared to 16N for untreated cells. NIC-treated cells also displayed dramatic differences in morphology - characterized by an increase in cell size, the presence of a more highly lobated nucleus, and an increased frequency of proplatelet-forming cells. Nicotinamide is known to inhibit poly(ADP-ribose) polymerase (PARP) and Sir2, which are both NAD+ dependent enzymes. Preliminary experiments show that PARP activity is low in cultured Mks and is not affected by addition of 6.25 mM NIC. Continued exposure (beginning at day 5) to the PARP inhibitors (and nicotinamide analogs) 3-aminobenzamide (3-AB) and benzamide at concentrations of 1, 3, and 6.25 mM was toxic to cells in a dose dependent manner. Interestingly, high doses of NIC (25 and 50 mM) were also toxic to cells. Remarkably, while Mk polyploidization and apoptosis are typically correlated, the increase in DNA content observed for NIC-treated cells occurred without significantly affecting the percentage of apoptotic Mks (assessed by Annexin V staining). These data suggest that it may be possible to partially decouple Mk apoptosis and polyploidization. Furthermore, while 6.25 mM NIC inhibited cell proliferation by ~35%, total expansion of cells cultured with 3 mM NIC was similar to that of untreated cells. This, combined with similar Mk commitment, as defined by a similar percentage of CD41+ cells, resulted in a greater overall number of high ploidy Mks in cultures treated with NIC. Since there is a direct correlation between Mk DNA content and platelet production (Mattia et al., Blood 99:888, 2002), these results suggest a possible therapeutic benefit of NIC for the management of thrombocytopenia. Similarly, NIC could also be used as an additive to ex vivo Mk cultures destined for transplantation. Figure Figure

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