Multiphoton and FLIM imaging in quantifying ex vivo and in vivo body organ kinetics of solutes

2020 ◽  
Author(s):  
Michael S. Roberts ◽  
Deborah Barkauskas ◽  
Haolu Wang ◽  
Xin Liu ◽  
Hauke Studier ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Body Organ ◽  
Kinetics Of

scholarly journals c-Myc inhibition is critical for HSC expansion and Rad51 expression

2018 ◽  
Author(s):  
Merve Aksoz ◽  
Esra Albayrak ◽  
Galip Servet Aslan ◽  
Raife Dilek Turan ◽  
Lamia Yazgi Alyazici ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Nos Inhibitor ◽  
Kinetics Of

c-Myc plays a major role in the maintenance of glycolytic metabolism and hematopoietic stem cell (HSC) quiescence. Targeting modulators of HSC quiescence and metabolism could lead to HSC cell cycle entry with concomitant expansion. Here we show that c-Myc inhibitor 10074-G5 treatment leads to 2-fold increase in murine LSKCD34low HSC compartment post 7 days. In addition, c-Myc inhibition increases CD34+ and CD133+ human HSC number. c-Myc inhibition leads to downregulation of glycolytic and cyclin-dependent kinase inhibitor (CDKI) gene expression ex vivo and in vivo. In addition, c-Myc inhibition upregulates major HDR modulator Rad51 expression in hematopoietic cells. Besides, c-Myc inhibition does not alter proliferation kinetics of endothelial cells, fibroblasts or adipose derived mesenchymal stem cells, however; it limits bone marrow derived mesenchymal stem cell proliferation. We further demonstrate that a cocktail of c-Myc inhibitor 10074-G5 along with tauroursodeoxycholic acid (TUDCA) and i-NOS inhibitor L-NIL provides a robust HSC maintenance and expansion ex vivo as evident by induction of all stem cell antigens analyzed. Intriguingly, the cocktail of c-Myc inhibitor 10074-G5, TUDCA and L-NIL improves HDR related gene expression. These findings provide tools to improve ex vivo HSC maintenance and expansion, autologous HSC transplantation and gene editing through modulation of HSC glycolytic and HDR pathways.

Neutrophil passage through isolated perfused rabbit lungs

1991 ◽  
Vol 261 (4) ◽  
pp. H1317-H1323
Author(s):  
H. Schutte ◽  
S. Rosseau ◽  
D. Walmrath ◽  
F. Grimminger ◽  
C. Ernst ◽  
...  
Keyword(s):  
Sodium Iodide ◽  
Ex Vivo ◽  
Venous Pressure ◽  
In Vivo Studies ◽  
Buffer Solution ◽  
Physiological Flow ◽  
Human Pmns ◽  
Isolated Lungs ◽  
Kinetics Of

Kinetics of polymorphonuclear leukocyte (PMN) lung passage were investigated in ex vivo isolated and ventilated left rabbit lungs, perfused with buffer solution at physiological flow rate. 111In-labeled PMNs of rabbit or human origin were injected into the pulmonary artery, and the first fraction of PMNs that rapidly passed the lung together with coinjected erythrocytes, was collected separately for external radioactivity counting. Washout of initially retained PMNs from the lung was monitored by use of a sodium-iodide detector. Recirculation of cells was avoided by insertion of a filter in the perfusion circuit. A fraction of 16.6 +/- 1.3% of rabbit PMNs rapidly passed the lung vasculature, followed by an exponential washout of initially retained PMNs [half-time (t50) of lung transit 8.1 +/- 0.6 min]. Slightly higher t50 (12.2 +/- 1.0 min) was obtained upon use of human PMNs. Reduction in flow by 50% caused a marked prolongation of PMN transit (t50 = 27.8 +/- 5.1 min), whereas increase in flow to 150% only insignificantly decreased t50. Rise in pulmonary venous pressure to 5 and 8 mmHg caused retardation of PMN lung transit (t50 = 15.3 +/- 0.6 and 31.6 +/- 3.6 min). Preincubation of PMNs with 2 ng/ml endotoxin for 1 h induced marked delay in PMN washout (t50 = 26.1 +/- 2.8 min). In conclusion, single-pass PMN kinetics in isolated lungs correspond to in vivo studies previously reported, thus allowing elucidation of PMN-endothelial interactions in an intact lung vasculature under standardized conditions.

c-Myc Inhibitor 10074-G5 Induces Murine and Human Hematopoietic Stem and Progenitor Cell Expansion and HDR Modulator Rad51 Expression

2019 ◽  
Vol 19 (6) ◽  
pp. 479-494 ◽  
Author(s):  
Merve Aksoz ◽  
Esra Albayrak ◽  
Galip Servet Aslan ◽  
Raife Dilek Turan ◽  
Lamia Yazgi Alyazici ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cell Cycle Entry ◽  
Nos Inhibitor ◽  
Kinetics Of

Background:c-Myc plays a major role in the maintenance of glycolytic metabolism and hematopoietic stem cell (HSC) quiescence.Objective:Targeting modulators of HSC quiescence and metabolism could lead to HSC cell cycle entry with concomitant expansion.Methods and Results:Here we show that c-Myc inhibitor 10074-G5 treatment leads to 2-fold increase in murine LSKCD34low HSC compartment post 7 days. In addition, c-Myc inhibition increases CD34+ and CD133+ human HSC number. c-Myc inhibition leads to downregulation of glycolytic and cyclindependent kinase inhibitor (CDKI) gene expression ex vivo and in vivo. In addition, c-Myc inhibition upregulates major HDR modulator Rad51 expression in hematopoietic cells. Besides, c-Myc inhibition does not alter proliferation kinetics of endothelial cells, fibroblasts or adipose-derived mesenchymal stem cells, however, it limits bone marrow derived mesenchymal stem cell proliferation. We further demonstrate that a cocktail of c-Myc inhibitor 10074-G5 along with tauroursodeoxycholic acid (TUDCA) and i-NOS inhibitor L-NIL provides a robust HSC maintenance and expansion ex vivo as evident by induction of all stem cell antigens analyzed. Intriguingly, the cocktail of c-Myc inhibitor 10074-G5, TUDCA and L-NIL improves HDR related gene expression.Conclusion:These findings provide tools to improve ex vivo HSC maintenance and expansion, autologous HSC transplantation and gene editing through modulation of HSC glycolytic and HDR pathways.

Pilot Study Of Regulatory T Cell Depletion In The Setting Of Autologous Stem Cell Transplantation For Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4607-4607 ◽  
Author(s):  
Gottfried von Keudell ◽  
Cara A Rosenbaum ◽  
Todd M. Zimmerman ◽  
Michael Bishop ◽  
Koen van Besien ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Stem Cell ◽  
Pilot Study ◽  
Stem Cell Transplantation ◽  
Ex Vivo ◽  
Treg Depletion ◽  
Before And After ◽  
Kinetics Of

Although autologous stem cell transplantation (ASCT) is effective for patients with multiple myeloma (MM), disease relapse remains problematic. Despite significant advances in therapeutics for MM, it remains mostly incurable. Following ASCT, a majority of MM patients are in a minimal residual disease state, at which time delivery of immunotherapy may be most effective. Regulatory T cells (Tregs) are a naturally-suppressive CD4+ T cell population (CD4+CD25+FoxP3+ T cells) that expand in MM patients, and strongly inhibit anti-tumor immune responses. In pre-clinical models Treg depletion enhanced the function of tumor antigen-specific T cells. Effective strategies to deplete Tregs in humans are being investigated. We initiated pilot study to test 2 methods of Treg depletion in MM patients undergoing ASCT. In the first approach, an anti-CD25 monoclonal antibody (basiliximab) was administered day +1 following ASCT (in vivo Treg depletion). The second method involved depleting CD4+CD25+ Tregs from autologous stem cell (ASC) grafts with clinical-grade anti-CD25 microbeads and the CliniMACS device (Miltenyi) (ex vivo Treg depletion). A control arm consisted of patients in which no Treg-depleting maneuver was performed. To date, 10 patients with symptomatic MM have been enrolled and randomly assigned to 1 of 3 study arms (arm 1 – standard ASCT; arm 2 – in vivo Treg depletion; arm 3 – ex vivo Treg depletion). Primary endpoints included: a) efficacy of ex vivo Treg depletion from ASC grafts, b) kinetics of Treg depletion and recovery in each study arm, and c) assessment of toxicity associated with Treg depletion. Secondary endpoints included time to engraftment following ASCT and disease response. The overall goal of the study was to identify a superior strategy with regard to depth and durability of Treg depletion. Ten patients (median age 59; range 46-68) have been enrolled. 4 were enrolled onto arm 1, 3 onto arm 2 and 3 onto arm 3. One patient enrolled on arm 2 was removed from study due to ASC mobilization failure. ASC were collected following neupogen and plexifor, and conditioning for ASCT consisted of melphalan (200mg/m2). Typical serious adverse events associated with ASCT were observed in all study arms, with neutropenic fever being the most common (n=2 arm 1, n=1 arm 2 and n=2 arm 3). Autoimmune complications, such as autologous graft-versus-host disease, were not observed. All patients engrafted post-ASCT with normal kinetics. Too few patients have been treated to make conclusions regarding clinical efficacy. Depletion of CD4+CD25+ Tregs from ASC grafts in arm 3 (ex vivo Treg depletion) was effective. As shown in Figure 1, 90.1% +/- 6% of CD4+25+ Tregs were depleted from 3 ASC grafts following the procedure. Baseline frequencies of peripheral blood (PB) CD4+FoxP3+ Tregs were similar between patients in all arms (p = n.s.). At least a 2-fold reduction in PB Treg frequency was observed at days +28 and + 60 following ASCT (Figure 2). At day +28, mean numbers (+/- SD) of CD4+FoxP3+ PB Tregs were 8.58% +/- 4.52%, 2.48% +/- 1.77%, and 3.28% +/- 1.12% in arms 1, 2 and 3, respectively (p = 0.08 arm 1 vs arm 2; p = 0.05 arm 1 vs arm 3; p = .57 arm 2 vs arm 3). These data suggest that both the in vivo and ex vivo Treg depletion may be effective at mediating effective and durable Treg depletion.Figure 1Treg are effectively depleted ex vivo with CD25 microbeads and the Clinimacs device. A FACS plots showing frequency of CD4 +CD25 + Tregs before (left) and after (right) ex vivo Treg depletion procedure (arm 3). B Mean CD4+CD25+ Treg frequencies before and after ex vivo Treg depletion (*p= 0.05).Figure 1. Treg are effectively depleted ex vivo with CD25 microbeads and the Clinimacs device. A FACS plots showing frequency of CD4 +CD25 + Tregs before (left) and after (right) ex vivo Treg depletion procedure (arm 3). B Mean CD4+CD25+ Treg frequencies before and after ex vivo Treg depletion (*p= 0.05).Figure 2Kinetics of Treg depletion following ASCT. Mean (+/- SD) frequency of CD4+FoxP3+ Tregs before and after ASCT in each study arm are displayed.Figure 2. Kinetics of Treg depletion following ASCT. Mean (+/- SD) frequency of CD4+FoxP3+ Tregs before and after ASCT in each study arm are displayed. In conclusion, both the in vivo or ex vivo method of Treg depletion appear to be effective based on small patient numbers. Treg depletion in MM patients undergoing ASCT is safe and does not delay engraftment. Additional patients will need to be treated to make conclusions regarding clinical efficacy. Future studies of Treg depletion in combination with other immunotherapeutic strategies are being considered. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ex Vivo and In Vivo Properties of an Injectable Hydrogel Derived From Acellular Ear Cartilage Extracellular Matrix

2021 ◽  
Vol 9 ◽  
Author(s):  
Danni Gong ◽  
Fei Yu ◽  
Meng Zhou ◽  
Wei Dong ◽  
Dan Yan ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Extracellular Matrix ◽  
Histological Analysis ◽  
Ex Vivo ◽  
Positive Staining ◽  
Gelation Kinetics ◽  
Ear Cartilage ◽  
Kinetics Of

Extracellular matrix (ECM) hydrogels provide advantages such as injectability, the ability to fill an irregularly shaped space, and the adequate bioactivity of native matrix. In this study, we developed decellularized cartilage ECM (dcECM) hydrogels from porcine ears innovatively via the main method of enzymatic digestion and verified good biocompatible properties of dcECM hydrogels to deliver chondrocytes and form subcutaneous cartilage in vivo. The scanning electron microscopy and turbidimetric gelation kinetics were used to characterize the material properties and gelation kinetics of the dcECM hydrogels. Then we evaluated the biocompatibility of hydrogels via the culture of chondrocytes in vitro. To further explore the dcECM hydrogels in vivo, grafts made from the mixture of dcECM hydrogels and chondrocytes were injected subcutaneously in nude mice for the gross and histological analysis. The structural and gelation kinetics of the dcECM hydrogels altered according to the variation in the ECM concentrations. The 10 mg/ml dcECM hydrogels could support the adhesion and proliferation of chondrocytes in vitro. In vivo, at 4 weeks after transplantation, cartilage-like tissues were detected in all groups with positive staining of toluidine blue, Safranin O, and collagen II, indicating the good gelation of dcECM hydrogels. While with the increasing concentration, the tissue engineering cartilages formed by 10 mg/ml dcECM hydrogel grafts were superior in weights, volumes, collagen, and glycosaminoglycan (GAG) content compared to the dcECM hydrogels of 1 mg/ml and 5 mg/ml. At 8 weeks after grafting, dcECM hydrogel grafts at 10 mg/ml showed very similar qualities to the control, collagen I grafts. After 12 weeks of in vivo culture, the histological analysis indicated that 10 mg/ml dcECM hydrogel grafts were similar to the normal cartilage from pig ears, which was the source tissue. In conclusion, dcECM hydrogel showed the promising potential as a tissue engineering biomaterial to improve the regeneration and heal injuries of ear cartilage.

Kinetics of Rat Skin Optical Clearing at Topical Application of 40%Glucose: Ex Vivo and In Vivo Studies

2019 ◽  
Vol 25 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Daria K. Tuchina ◽  
Polina A. Timoshina ◽  
Valery V. Tuchin ◽  
Alexey N. Bashkatov ◽  
Elina A. Genina
Keyword(s):  
Topical Application ◽  
Ex Vivo ◽  
Rat Skin ◽  
Optical Clearing ◽  
Kinetics Of

scholarly journals Comparative characterization of the reassortant Orthobunyavirus Ngari with putative parental viruses, Bunyamwera and Batai: in vitro characterization and ex vivo stability

2020 ◽  
Author(s):  
M. Fausta Dutuze ◽  
E. Handly Mayton ◽  
Joshua D. Macaluso ◽  
Rebecca C. Christofferson
Keyword(s):  
Ex Vivo ◽  
Laboratory System ◽  
In Vitro Characterization ◽  
Significant Difference ◽  
The Relationship ◽  
Kinetics Of ◽  
Negative Sense

Bunyamwera (BUNV), Batai (BATV) and Ngari (NRIV) are mosquito-borne viruses that are members of the genus Orthobunyavirus in the order Bunyavirales. These three viruses are enveloped with single-stranded, negative-sense RNA genomes consiting of three segments, denoted as Small (S), Medium (M) and Large (L). Ngari is thought to be the natural reassortant progeny of Bunyamwera and Batai viruses. The relationship between these ‘parental’ viruses and the ‘progeny’ poses an interesting question, especially given that there is overlap in their respective transmission ecologies, but differences in their infection host ranges and pathogenesis. We compared the in vivo kinetics of these three viruses in a common laboratory system and found no significant difference in growth kinetics. There was, however, a tendency of BATV to have smaller plaques than either BUNV or NRIV. Furthermore, we determined that all three viruses are stable in extracellular conditions and retain infectivity for a week in non-cellular media, which has public health and biosafety implications. The study of this understudied group of viruses addresses a need for basic characterization of viruses that have not yet reached epidemic transmission intensity, but that have the potential due to their infectivity to both human and animal hosts. These results lay the groundwork for future studies of these neglected viruses of potential public and One Health importance.

scholarly journals Cell type-specific and time-dependent light exposure contribute to silencing in neurons expressing Channelrhodopsin-2

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Alexander M Herman ◽  
Longwen Huang ◽  
Dona K Murphey ◽  
Isabella Garcia ◽  
Benjamin R Arenkiel
Keyword(s):  
Ex Vivo ◽  
Light Exposure ◽  
Cell Types ◽  
Specific Cell ◽  
Depolarization Block ◽  
Light Pulses ◽  
Cell Type Specific ◽  
Photo Response ◽  
Kinetics Of

Channelrhodopsin-2 (ChR2) has quickly gained popularity as a powerful tool for eliciting genetically targeted neuronal activation. However, little has been reported on the response kinetics of optogenetic stimulation across different neuronal subtypes. With excess stimulation, neurons can be driven into depolarization block, a state where they cease to fire action potentials. Herein, we demonstrate that light-induced depolarization block in neurons expressing ChR2 poses experimental challenges for stable activation of specific cell types and may confound interpretation of experiments when ‘activated’ neurons are in fact being functionally silenced. We show both ex vivo and in vivo that certain neuronal subtypes targeted for ChR2 expression become increasingly susceptible to depolarization block as the duration of light pulses are increased. We find that interneuron populations have a greater susceptibility to this effect than principal excitatory neurons, which are more resistant to light-induced depolarization block. Our results highlight the need to empirically determine the photo-response properties of targeted neurons when using ChR2, particularly in studies designed to elicit complex circuit responses in vivo where neuronal activity will not be recorded simultaneous to light stimulation.

scholarly journals Bright flash response recovery of mammalian rods in vivo is rate limited by RGS9

2017 ◽  
Vol 149 (4) ◽  
pp. 443-454 ◽  
Author(s):  
Gabriel Peinado Allina ◽  
Christopher Fortenbach ◽  
Franklin Naarendorp ◽  
Owen P. Gross ◽  
Edward N. Pugh ◽  
...  
Keyword(s):  
G Protein ◽  
Wheel Running ◽  
Ex Vivo ◽  
Scotopic Vision ◽  
Bright Flash ◽  
Wide Range ◽  
Genetic Perturbations ◽  
Rate Limiting ◽  
Kinetics Of

The temporal resolution of scotopic vision is thought to be constrained by the signaling kinetics of retinal rods, which use a highly amplified G-protein cascade to transduce absorbed photons into changes in membrane potential. Much is known about the biochemical mechanisms that determine the kinetics of rod responses ex vivo, but the rate-limiting mechanisms in vivo are unknown. Using paired flash electroretinograms with improved signal-to-noise, we have recorded the amplitude and kinetics of rod responses to a wide range of flash strengths from living mice. Bright rod responses in vivo recovered nearly twice as fast as all previous recordings, although the kinetic consequences of genetic perturbations previously studied ex vivo were qualitatively similar. In vivo, the dominant time constant of recovery from bright flashes was dramatically reduced by overexpression of the RGS9 complex, revealing G-protein deactivation to be rate limiting for recovery. However, unlike previous ex vivo recordings, dim flash responses in vivo were relatively unaffected by RGS9 overexpression, suggesting that other mechanisms, such as calcium feedback dynamics that are strongly regulated by the restricted subretinal microenvironment, act to determine rod dim flash kinetics. To assess the consequences for scotopic vision, we used a nocturnal wheel-running assay to measure the ability of wild-type and RGS9-overexpressing mice to detect dim flickering stimuli and found no improvement when rod recovery was speeded by RGS9 overexpression. These results are important for understanding retinal circuitry, in particular as modeled in the large literature that addresses the relationship between the kinetics and sensitivity of retinal responses and visual perception.

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