Rapid, Exponential Extra-Splenic Platelet Consumption in WASP Deficient Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2174-2174
Author(s):  
Ted S. Strom ◽  
Jim Y. Wan ◽  
Haiming Du ◽  
Carl W. Jackson
Keyword(s):  
Platelet Count ◽  
Consumption Rate ◽  
Ex Vivo ◽  
Fold Increase ◽  
Extramedullary Hematopoiesis ◽  
Plateau Phase ◽  
Platelet Production ◽  
Normal Platelet ◽  
Deficient Mice

Abstract The thrombocytopenia seen in patients with the Wiskott-Aldrich Syndrome (WAS) is thought to be due primarily to rapid platelet consumption, and is markedly improved by splenectomy. While a murine model of WAS shows only mild thrombocytopenia, we have previously demonstrated rapid platelet turnover in this model; that splenectomy improves the platelet count in WASP-(C57Bl/6J) mice; and that the latter mice do not achieve the same platelet count found in splenectomized WT mice. Here we show that CMFDA-labeled WASP- platelets are consumed rapidly and exponentially in WT recipients, with an estimated lifespan of 18 hours (vs. 87 hours for WT platelets). WASP- platelets are consumed only slightly more slowly in splenectomized WT recipients (lifespan 28 hours, vs. 102 hr for WT platelets). On the C3H background, WASP- mice have normal platelet counts but show a similar rapid, exponential platelet consumption rate (lifespan 25 hours in either C3H or C57Bl/6J recipients, vs 85 hours for WT platelets). In vivo platelet biotinylation studies demonstrate less effective labeling of WASP- (C57Bl/6J) platelets than of WT platelets. After a plateau phase, in vivo labeled WASP- platelets show the same kind of rapid platelet turnover seen with ex vivo labeling (figure 1). Our results imply a three-fold increase in platelet production rate in WASP- mice on either background, consistent with the markedly increased splenic extramedullary hematopoiesis seen in WASP- mice. WASP- mice also show an increased number of bone marrow megkaryocytes, the ploidy distribution of which is normal. We conclude that WASP- mice demonstrate a significantly increased rate of extra-splenic platelet consumption that is largely (on the C57Bl/6 background) or completely (on the C3H background) compensated by increased platelet production. The ability of WASP- mice to compensate for their rapid platelet consumption, and the normal ploidy of their megakaryocytes, suggests that platelet production is not impaired by WASP deficiency in this model. In vivo biotinylation of WASP-and WT platelets In vivo biotinylation of WASP-and WT platelets

Impaired “outside-in” integrin αIIbβ3 signaling and thrombus stability in TSSC6-deficient mice

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 1911-1918 ◽  
Author(s):  
Matt W. Goschnick ◽  
Lai-Man Lau ◽  
Janet L. Wee ◽  
Yong S. Liu ◽  
P. Mark Hogarth ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Fluorescein Isothiocyanate ◽  
Clot Retraction ◽  
In Vivo Evaluation ◽  
Integrin Αiibβ3 ◽  
Normal Platelet ◽  
Granule Secretion ◽  
Platelet Spreading ◽  
Deficient Mice

AbstractWe investigated the role of the hematopoietic-specific tetraspanin superfamily member, TSSC6, in platelet function using wild-type mice and TSSC6-deficient mice. TSSC6 is expressed on the surface of murine platelets and is up-regulated by thrombin stimulation, indicating an intracellular pool of TSSC6. Immunoprecipitation/Western blot studies reveal a constitutive physical association of TSSC6 with the integrin αIIbβ3 complex under strong detergent conditions. In vivo evaluation of hemostasis by tail bleeding revealed increased bleeding time, volume of blood lost, and evidence of tail rebleeds in TSSC6 null mice, indicating unstable hemostasis. Using ex vivo techniques, we showed that TSSC6-deficient platelets exhibited impaired kinetics of clot retraction, platelet aggregation at lower doses of PAR-4, and collagen and platelet spreading on fibrinogen in the presence of normal integrin αIIbβ3 expression. TSSC6-deficient platelets showed normal alpha granule secretion, normal “insideout” integrin αIIbβ3 signaling (fluorescein isothiocyanate [FITC]–fibrinogen and JON/A binding), and normal platelet adhesion on fibrinogen. Furthermore, we show that absence of platelet TSSC6 affects the secondary stability of arterial thrombi in vivo upon vascular injury. These data demonstrate that TSSC6 appears to regulate integrin αIIbβ3 “outside-in” signaling events in platelets and is necessary for stability of arterial thrombi in vivo.

scholarly journals The Y14-p53 Regulatory Circuit in Megakaryocyte Differentiation and Thrombocytopenia

2020 ◽  
Author(s):  
Chun-Hao Su ◽  
Wei-Ju Liao ◽  
Wei-Chi Ke ◽  
Ruey-Bing Yang ◽  
Woan-Yuh Tarn
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Ex Vivo ◽  
Critical Role ◽  
Platelet Production ◽  
Noncoding Regions ◽  
Regulatory Circuit ◽  
Hel Cells ◽  
Absent Radius ◽  
Megakaryocyte Differentiation

SUMMARYThrombocytopenia-absent radius syndrome is caused by a deletion in chromosome 1q21.1 in trans with RBM8A mutations in the noncoding regions. We generated megakaryocyte-specific Rbm8a knockout (Rbm8aKOMK) mice that exhibited marked thrombocytopenia, internal hemorrhage, and splenomegaly, indicating a disorder of platelet production. Rbm8aKOMK mice accumulated immature megakaryocytes in the bone marrow and spleen. Depletion of Y14/RBM8A in human erythroleukemia (HEL) cells inhibited phorbol ester-induced polyploidy and downregulated the signaling pathways associated with megakaryocyte maturation. Accordingly, Rbm8aKOMK mice had reduced expression of surface glycoproteins on platelets and impaired coagulation. Moreover, p53 level was increased in Y14-depleted HEL cells and Rbm8aKOMK megakaryocytes. Treatment with a p53 inhibitor restored ex vivo differentiation of Rbm8aKOMK megakaryocytes and unexpectedly activated Y14 expression in HEL cells. Knockout of Trp53 in part restored the platelet count of Rbm8aKOMK mice. These results indicate that the Y14-p53 circuit plays a critical role in megakaryocyte differentiation and platelet production.

scholarly journals Augmented expansion of Treg cells from healthy and autoimmune subjects via adult progenitor cell co-culture

2020 ◽  
Author(s):  
JL Reading ◽  
VD Roobrouck ◽  
CM Hull ◽  
PD Becker ◽  
J Beyens ◽  
...  
Keyword(s):  
T Cell ◽  
Treg Cell ◽  
Regulatory T Cell ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Fold Increase ◽  
Adoptive Cellular Therapy ◽  
T Cell Therapy

AbstractRecent clinical experience has demonstrated that adoptive regulatory T cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote regulatory T cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a GMP compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is linked with a distinct Treg cell-intrinsic transcriptional program, characterized by diminished levels of core exhaustion (BATF, ID2, PRDM1, LAYN, DUSP1), and quiescence (TOB1, TSC22D3) related genes, coupled to elevated expression of cell-cycle and proliferation loci (MKI67, CDK1, AURKA, AURKB). In addition, MulTreg display a unique gut homing (CCR7lo β7hi) phenotype and importantly, are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or Th1-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 TSDR demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno graft vs host disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

scholarly journals Regulation of megakaryocyte ploidy in vivo in the rat

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 74-81 ◽  
Author(s):  
DJ Kuter ◽  
RD Rosenberg
Keyword(s):  
Platelet Count ◽  
Feedback Loop ◽  
Antiplatelet Antibody ◽  
Normal Platelet Count ◽  
Normal Platelet ◽  
Plasma Factor ◽  
Ploidy Changes ◽  
The Relationship ◽  
Made In

Abstract The relationship between the bone marrow (BM) megakaryocyte and the circulating platelet was explored. Incremental changes in platelet count were made in rats by infusion of antiplatelet antibody or by platelet transfusion, and the response of megakaryocytes was measured by flow cytometry. Proportional changes in megakaryocyte ploidy were demonstrated: As the platelet count declined, ploidy increased; as the platelet count increased, ploidy decreased. Even moderate degrees of thrombocytopenia and thrombocytosis (48% and 177% of the normal platelet count) were associated with changes in ploidy. These changes were not the results of the technique used to alter the platelet count because reinfusion of platelets after 3 hours of thrombocytopenia prevented any ploidy change. These studies proved that the circulating platelet and the megakaryocyte constitute a classic feedback loop whose activity can be measured by the degree of ploidization of the megakaryocyte. The minimal duration of thrombocytopenia necessary to promote megakaryocyte ploidy changes was approximately 10 hours. Using a BM culture assay, we identified a plasma factor which induced alterations in megakaryocyte ploidy and whose level is inversely proportional to the platelet count.

scholarly journals 2215

2017 ◽  
Vol 1 (S1) ◽  
pp. 3-3
Author(s):  
Timothy P. Moran ◽  
Robert M. Immormino ◽  
Hideki Nakano ◽  
David Peden ◽  
Donald N. Cook
Keyword(s):  
Flow Cytometry ◽  
Airway Inflammation ◽  
Allergic Asthma ◽  
Immune Cells ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Allergic Airway Inflammation ◽  
Surface Protein ◽  
Fold Increase ◽  
Deficient Mice

OBJECTIVES/SPECIFIC AIMS: Allergic asthma is a chronic lung disease driven by inappropriate inflammatory responses against inhaled allergens. Neuropilin-2 (NRP2) is a pleiotropic transmembrane receptor expressed in the lung, but its role in allergic airway inflammation is unknown. Here, we characterized NRP2 expression in lung immune cells and investigated the effects of NRP2 deficiency on airway inflammation. METHODS/STUDY POPULATION: NRP2 expression by lung immune cells from NRP2 reporter mice was determined by flow cytometry. NRP2 expression by human alveolar macrophages (AM) from healthy individuals was determined by mRNA analysis and flow cytometry. Airway inflammation in NRP2-deficient mice was assessed by bronchoalveolar lavage (BAL) cytology and inflammatory gene expression in lung tissue. RESULTS/ANTICIPATED RESULTS: NRP2 expression in lung immune cells was negligible under steady-state conditions. In contrast, inhalational exposure to lipopolysaccharide (LPS) adjuvant dramatically induced NRP2 expression in AM, as 63.3% of AM from LPS-treated mice were NRP2+ compared with 1.5% of AM from control mice. Ex vivo treatment of human AM with LPS resulted in a 1.5-fold and 2.6-fold increase in NRP2 mRNA and surface protein expression, respectively. Compared to littermate controls, NRP2-deficient mice had greater numbers of BAL leukocytes and increased lung expression of the T helper type 2 cytokines IL-4 and IL-5. Furthermore, NRP2 deficiency resulted in stochastic development of allergic airway inflammation, as spontaneous airway eosinophilia was detected in 25% (2/8) of NRP2-deficient mice compared with 0% (0/8) of littermate controls. DISCUSSION/SIGNIFICANCE OF IMPACT: NRP2 is expressed by activated human and murine AM and suppresses the spontaneous development of allergic airway inflammation. These findings suggest that NRP2 may play a key role in allergic asthma pathogenesis, and could prove to be an important therapeutic target in patients with asthma and other allergic diseases.

scholarly journals RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival

Blood ◽  
2020 ◽  
Vol 136 (15) ◽  
pp. 1773-1782 ◽  
Author(s):  
Daniel DeHelian ◽  
Shuchi Gupta ◽  
Jie Wu ◽  
Chelsea Thorsheim ◽  
Brian Estevez ◽  
...  
Keyword(s):  
Platelet Count ◽  
G Protein ◽  
Platelet Activation ◽  
Platelet Reactivity ◽  
Rgs Proteins ◽  
Platelet Recovery ◽  
Platelet Production ◽  
Platelet Survival

Abstract G protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10−/− platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18−/− mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18−/− and RGS10−/−18−/− mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

scholarly journals In vivo adenovirus vector-mediated transfer of the human thrombopoietin cDNA maintains platelet levels during radiation-and chemotherapy- induced bone marrow suppression

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 778-784 ◽  
Author(s):  
A Ohwada ◽  
S Rafii ◽  
MA Moore ◽  
RG Crystal
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Adenovirus Vector ◽  
Bone Marrow Suppression ◽  
Single Administration ◽  
Platelet Recovery ◽  
Platelet Production ◽  
Life Threatening ◽  
Bone Abnormalities

Abstract Thrombopoietin (TPO, c-mpl ligand) has emerged as a major hematopoietic cytokine stimulating megakaryocyte proliferation, endomitosis, and platelet production. This study shows that a single administration of an adenovirus (Ad) vector encoding TPO (AdCMV.TPO) abrogates thrombocytopenia induced in mice by carboplatin and irradiation. Normal Balb/c mice receiving the vector had increased platelet counts peaking at 7 days and returning to baseline by day 15. Mice rendered pancytopenic with 500 rads and 1.2 mg of carboplatin had a nadir platelet count of five percent of the baseline. Mice receiving AdCMV.TPO 3 days before receiving irradiation and chemotherapy achieved a platelet nadir fourfold higher, and had significant reduction in duration of thrombocytopenia, than mice receiving the control Ad vector. Introduction of AdCMV.TPO the same day of chemotherapy and irradiation was equally effective in acceleration of platelet recovery, but administration of AdCMV.TPO 3 days after chemotherapy-radiation had little effect on platelet recovery. At 30 days after therapy bone marrow and spleen of mice treated with AdCMV.TPO were populated with a large number of polyploid megakaryocytes, but there was no evidence of circulating megakaryocytes in the liver or lungs and no pathologic bone abnormalities such as osteosclerosis or myelofibrosis. These observations suggest that an Ad vector may be an excellent delivery system to provide adequate TPO production to maintain platelet levels in circumstances associated with life-threatening thrombocytopenia.

scholarly journals Subcellular distribution of selenium in deficient mouse liver

1989 ◽  
Vol 258 (2) ◽  
pp. 541-545 ◽  
Author(s):  
R Reiter ◽  
R Otter ◽  
A Wendel
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Trace Element ◽  
Fold Increase ◽  
Subcellular Fractionation ◽  
Fast Response ◽  
Cell Compartment ◽  
Deficient Mice

Selenium (Se)-deficient mice were labelled in vivo with single pulses of [75Se]selenite, and the intrahepatic distribution of the trace element was studied by subcellular fractionation. At 1 h after intraperitoneal injection of 3.3 or 10 micrograms of Se/kg body weight, 15% of the respective doses were found in the liver. Accumulation in the subcellular fractions followed the order: Golgi vesicular much greater than lysosomal greater than cytosolic = microsomal greater than mitochondrial, peroxisomal, nuclear and plasma-membrane fraction. At a dose of 3.3 micrograms/kg, more than 90% of the hepatic Se was protein-bound. When cross-contamination was accounted for, the following specific Se contents of the subcellular compartments were extrapolated: Golgi apparatus, 7.50 pmol/mg; cytosol, 0.90 pmol/mg; endoplasmic reticulum, 0.80 pmol/mg; mitochondria, 0.49 pmol/mg; nuclei, lysosomes, peroxisomes and plasma membrane, less than 0.4 pmol/mg. At 10 micrograms/kg, a roughly 2-3-fold increase in Se content of all fractions was found without major changes in the intrahepatic distribution pattern. An extraordinary rise in the cytosolic fraction was due to an apparently non-protein-bound Se pool. At 24 h after dosing, total hepatic Se had decreased to 6% of the initial dose and had become predominantly protein-bound. The 60% decrease in hepatic Se was reflected in a similar fall in the subcellular levels of the trace element. The Golgi apparatus still had the highest specific Se content, although accumulation was 5 times less than that after 1 h. The cytosolic pool accounted for 50% of the hepatic Se at both labelling times. After 1 h the Golgi apparatus was, with 19%, the second largest intrahepatic pool, followed by the endoplasmic reticulum with 16%. The high affinity and fast response of the Golgi apparatus to Se supplementation of deficient mice is interpreted in terms of a predominant function of this cell compartment in the processing and the export of Se-proteins from the liver.

scholarly journals Nanocrystals of Fusidic Acid for Dual Enhancement of Dermal Delivery and Antibacterial Activity: In Vitro, Ex Vivo and In Vivo Evaluation

Pharmaceutics ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 199 ◽  
Author(s):  
Iman S. Ahmed ◽  
Osama S. Elnahas ◽  
Nouran H. Assar ◽  
Amany M. Gad ◽  
Rania El Hosary
Keyword(s):  
Fusidic Acid ◽  
Therapeutic Efficacy ◽  
Ex Vivo ◽  
Skin Permeation ◽  
Fold Increase ◽  
Spherical Particles ◽  
Resistant Bacteria ◽  
Infection Model

With the alarming rise in incidence of antibiotic-resistant bacteria and the scarcity of newly developed antibiotics, it is imperative that we design more effective formulations for already marketed antimicrobial agents. Fusidic acid (FA), one of the most widely used antibiotics in the topical treatment of several skin and eye infections, suffers from poor water-solubility, sub-optimal therapeutic efficacy, and a significant rise in FA-resistant Staphylococcus aureus (FRSA). In this work, the physico-chemical characteristics of FA were modified by nanocrystallization and lyophilization to improve its therapeutic efficacy through the dermal route. FA-nanocrystals (NC) were prepared using a modified nanoprecipitation technique and the influence of several formulation/process variables on the prepared FA-NC characteristics were optimized using full factorial statistical design. The optimized FA-NC formulation was evaluated before and after lyophilization by several in-vitro, ex-vivo, and microbiological tests. Furthermore, the lyophilized FA-NC formulation was incorporated into a cream product and its topical antibacterial efficacy was assessed in vivo using a rat excision wound infection model. Surface morphology of optimized FA-NC showed spherical particles with a mean particle size of 115 nm, span value of 1.6 and zeta potential of −11.6 mV. Differential scanning calorimetry and powder X-ray diffractometry confirmed the crystallinity of FA following nanocrystallization and lyophilization. In-vitro results showed a 10-fold increase in the saturation solubility of FA-NC while ex-vivo skin permeation studies showed a 2-fold increase in FA dermal deposition from FA-NC compared to coarse FA. Microbiological studies revealed a 4-fofd decrease in the MIC against S. aureus and S. epidermidis from FA-NC cream compared to commercial Fucidin cream. In-vivo results showed that FA-NC cream improved FA distribution and enhanced bacterial exposure in the infected wound, resulting in increased therapeutic efficacy when compared to coarse FA marketed as Fucidin cream.

scholarly journals Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies

2017 ◽  
Vol 312 (1) ◽  
pp. E27-E36 ◽  
Author(s):  
Servane Le Plénier ◽  
Arthur Goron ◽  
Athanassia Sotiropoulos ◽  
Eliane Archambault ◽  
Chantal Guihenneuc ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ex Vivo ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Fold Increase ◽  
Underlying Mechanism ◽  
Muscle Homeostasis

Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated ( P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control.

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