Microvesicles Bind Soluble Fibrinogen, Adhere to Immobilized Fibrinogen and Coaggregate with Platelets

1998 ◽  
Vol 79 (02) ◽  
pp. 389-394 ◽  
Author(s):  
Nils Olav Solum ◽  
Frank Brosstad ◽  
Turid Pedersen ◽  
Marit Kveine ◽  
Pål André Holme
Keyword(s):  
Flow Cytometry ◽  
Alkaline Phosphatase ◽  
Receptor Complex ◽  
Binding Studies ◽  
Stimulation Of

SummaryIn the present study we have investigated whether platelet derived microvesicles can bind soluble fibrinogen, bind to immobilized fibrinogen, and coaggregate with platelets. Flow cytometry was used for studies on binding of soluble fibrinogen and coaggregation, whereas ELISA wells were used to study binding of microvesicles to immobilized fibrinogen. Biotinylated microvesicles produced by stimulation with A23187, thrombin or SFLLRN of platelets which had been surface-labelled with biotin, were used both for the coaggregation experiments and for the binding studies with immobilized fibrinogen. Unlabelled microvesicles and biotinylated fibrinogen were employed when studying binding of soluble fibrinogen to the microvesicles. For the flow cytometry, the biotinylated proteins were reacted with avidin or streptavidin which was PE-conjugated, whereas the same substances were conjugated with alkaline phosphatase for the ELISA studies. The microvesicles formed after stimulation of platelets by SFLLRN or A23187 clearly bound the soluble, biotinylated fibrinogen. Moreover, isolated biotinylated microvesicles added to washed platelets prior to activation, were associated to the microaggregates that formed after stimulation. A significant binding of biotinylated microvesicles to immobilized fibrinogen could also be detected. The binding of micro-vesicles to soluble and immobilized fibrinogen and association to platelets was clearly specific and at least partly dependent on the GPIIb-IIIa complex, as all of these phenomena could be prevented or reduced by addition of the c7E3 Fab which blocks the activated form of this receptor complex. From these in vitro results it is clear that microvesicles can bind to immobilized fibrinogen, bind soluble fibrinogen and are able to coaggregate with platelets. It may be speculated that these results also reflect a haemostatic role of microvesicles in vivo.

scholarly journals The Role of Monocytes/Macrophages In The Pathogenesis of Immune Associated Diffuse Alveolar Hemorrhage

2021 ◽  
Author(s):  
Qing Wei ◽  
Xun Chen ◽  
Jing Liu ◽  
Yan Li ◽  
Guangmin Nong
Keyword(s):  
Flow Cytometry ◽  
Critical Role ◽  
Lavage Fluid ◽  
Peripheral Blood Monocyte ◽  
Healthy Children ◽  
Group 3 ◽  
Group 2 ◽  
Stimulation Of

Abstract Backgroud The studies in the immnue associated diffuse alveolar hemorrahge (DAH) animal models showed that monocytes/macrophages played an critical role in the pathogenesis.Whether monocytes/macrophages contribute to the pathogenesis of immune associated DAH in human is still unknow. The aim of this study was to explore the role of monocytes/macrophages in the pathogenesis of immune associated DAH in human.Methods This study was conducted in two parts. In the first part, 37 children with immune associated DAH were included (DAH group), and 18 healthy children were recruited as the controls (HC group). Peripheral blood monocyte subtype was analyzed using flow cytometry. In the second part, 24 children with immune associated DAH were included (DAH group), and 13 children with acute airway foreingn body or mild benign airway stenosis were included as the controls (HC group). Bronochoalveolar lavage fluid (BALF) was collected using bronchoscope. Cytokines in the BALF supernatant were detected using cytometric bread array. BALF supertanant was used to stimulated the macrophages in vitro. The mRNA relative expressions of IL-1β, TNFα, IL-6, TGM2, CD163 and MRC1 were detected using quantitative real-time PCR, and the expressions of CD14, CD80, CD86, CD163 and CD206 were detected using flow cytometry. Results 1. The percentage of classical monocyte was significantly increased, whereas the percentages of intermediate and non-classical monocyte were significantly decreased in the DAH group, when compared to those in the HC group. 2. The levels of MCP-1, IL-6 and IL-8 were all significantly higher in the BALF supernatant from the DAH group, when compared to those form the HC group. 3. The mRNA relative expressions of IL-1β and IL-6 as well as the expression of CD86 were significantly higher, whereas the mRNA relative expression of MRC1 as well as the expressions of CD163 and CD206 were significantly lower under the stimulation of BALF supernatant from the DAH group, when compared to that from the HC group. Conclusions Monocytes/macrophages might participate in the pathogenesis of immune associated DAH in human by enhanced M1 polarization.

Possible role of cyclic GMP in stimulus-secretion coupling by salt gland of the duck

1979 ◽  
Vol 237 (5) ◽  
pp. C200-C204 ◽  
Author(s):  
D. J. Stewart ◽  
J. Sax ◽  
R. Funk ◽  
A. K. Sen
Keyword(s):  
Cyclic Amp ◽  
Guanylate Cyclase ◽  
Cyclic Gmp ◽  
Salt Gland ◽  
Domestic Ducks ◽  
Stimulus Secretion Coupling ◽  
Stimulation Of

Stimulation of salt galnd secretion in domestic ducks in vivo increased the cyclic GMP concentration of the tissue, but had no effect on cyclic AMP levels. Methacholine, which is known to stimulate sodium transport by the glands both in vivo and in vitro, stimulated ouabain-sensitive respiration in salt gland slices. Cyclic GMP stimulated ouabain-sensitive respiration to the same extent as methacholine. Guanylate cyclase stimulators, hydroxylamine and sodium azide, also stimulated ouabain-sensitive respiration. The stimulation of ouabain-sensitive respiration by methacholine was blocked either by atropine or by removal of calcium from the incubation medium. The stimulation of ouabain-sensitive respiration by cyclic GMP still occurred in the absence of calcium. The above observations seem to indicate that cyclic GMP acts as a tertiary link in the process of stimulus-secretion coupling in the tissue.

Polar head groups are important for barrier-protective effects of oxidized phospholipids on pulmonary endothelium

2007 ◽  
Vol 292 (4) ◽  
pp. L924-L935 ◽  
Author(s):  
Anna A. Birukova ◽  
Panfeng Fu ◽  
Santipongse Chatchavalvanich ◽  
Dylan Burdette ◽  
Olga Oskolkova ◽  
...  
Keyword(s):  
Protective Effects ◽  
Oxidized Phospholipids ◽  
Barrier Dysfunction ◽  
Polar Head ◽  
Cell Counts ◽  
Head Groups ◽  
Stimulation Of

We have previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cell (EC) barrier function and demonstrated the critical role of cyclopentenone-containing modifications of arachidonoyl moiety in OxPAPC protective effects. In this study we used oxidized phosphocholine (OxPAPC), phosphoserine (OxPAPS), and glycerophosphate (OxPAPA) to investigate the role of polar head groups in EC barrier-protective responses to oxidized phospholipids (OxPLs). OxPAPC and OxPAPS induced sustained barrier enhancement in pulmonary EC, whereas OxPAPA caused a transient protective response as judged by measurements of transendothelial electrical resistance (TER). Non-OxPLs showed no effects on TER levels. All three OxPLs caused enhancement of peripheral EC actin cytoskeleton. OxPAPC and OxPAPS completely abolished LPS-induced EC hyperpermeability in vitro, whereas OxPAPA showed only a partial protective effect. In vivo, intravenous injection of OxPAPS or OxPAPC (1.5 mg/kg) markedly attenuated increases in the protein content, cell counts, and myeloperoxidase activities detected in bronchoalveolar lavage fluid upon intratracheal LPS instillation in mice, although OxPAPC showed less potency. All three OxPLs partially attenuated EC barrier dysfunction induced by IL-6 and thrombin. Their protective effects against thrombin-induced EC barrier dysfunction were linked to the attenuation of the thrombin-induced Rho pathway of EC hyperpermeability and stimulation of Rac-mediated mechanisms of EC barrier recovery. These results demonstrate for the first time the essential role of polar OxPL groups in blunting the LPS-induced EC dysfunction in vitro and in vivo and suggest the mechanism of agonist-induced hyperpermeability attenuation by OxPLs via reduction of Rho and stimulation of Rac signaling.

scholarly journals Cytoplasmic Dynein Nucleates Microtubules to Organize Them into Radial Arrays In Vivo

2004 ◽  
Vol 15 (6) ◽  
pp. 2742-2749 ◽  
Author(s):  
Viacheslav Malikov ◽  
Anna Kashina ◽  
Vladimir Rodionov
Keyword(s):  
Cytoplasmic Dynein ◽  
Exact Function ◽  
Necessary And Sufficient ◽  
Stimulation Of ◽  
In Cells

Numerous evidence demonstrates that dynein is crucial for organization of microtubules (MTs) into radial arrays, but its exact function in this process is unclear. Here, we studied the role of cytoplasmic dynein in MT radial array formation in the absence of the centrosome. We found that dynein is a potent MT nucleator in vitro and that stimulation of dynein activity in cytoplasmic fragments of melanophores induces nucleation-dependent formation of MT radial array in the absence of the centrosome. This new property of dynein, in combination with its known role as an MT motor that is essential for MT array organization in the absence and presence of the centrosome, makes it a unique molecule whose activity is necessary and sufficient for the formation and maintenance of MT radial arrays in cells.

Role of Il-2, Il-7, and IL-15 in T Cell Mediated Graft-vs-Leukemia Against Human Acute Lymphoblastic Leukemia in a NOD/scid Chimeric Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5256-5256
Author(s):  
Doug Cipkala ◽  
Kelly McQuown ◽  
Lindsay Hendey ◽  
Michael Boyer
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Mouse Model ◽  
Scid Mouse ◽  
In Vitro Cytotoxicity ◽  
Scid Mouse Model

Abstract The use of cytotoxic T-lymphocytes (CTL) has been attempted experimentally with various tumors to achieve disease control. Factors that may influence GVT include CTL cytotoxicity, ability to home to disease sites, and survival of T cells in the host. The objective of our study is to evaluate the GVL effects of human alloreactive CTL against ALL in a chimeric NOD/scid mouse model. CTL were generated from random blood donor PBMCs stimulated with the 697 human ALL cell line and supplemented with IL-2, -7, or -15. CTL were analyzed for in vitro cytotoxicity against 697 cells, phenotype, and in vitro migration on day 14. NOD/scid mice were injected with 107 697 ALL cells followed by 5x106 CTL. Mice were sacrificed seven days following CTL injection and residual leukemia was measured in the bone marrow and spleen via flow cytometry. The ratios of CD8/CD4 positive T cells at the time of injection were 46/21% for IL-2, 52/31% for IL-7, and 45/14% for IL-15 cultured CTL (n=13). Control mice not receiving CTL had a baseline leukemia burden of 2.01% and 0.15% in the bone marrow and spleen, respectively (n=15). Mice treated with IL-15 cultured CTL had a reduction in tumor burden to 0.2% (n=13, p=0.01) and 0.05% (n=13, p=0.01) in bone marrow and spleen, respectively. Those treated with IL-2 or IL-7 cultured CTL showed no significant difference in leukemia burden in either the bone marrow (IL-2 1.28%, Il-7 5.97%) or spleen (IL-2 0.4%, IL-7 0.33%). No residual CTL could be identified in the bone marrow or spleen at the time of sacrifice in any CTL group. CTL grown in each cytokine resulted in similar in vitro cytotoxicity at an effector:target ratio of 10:1 (IL-2 41.3%, IL-7 37.7%, IL-15 45.3%, n=12–15, p>0.05 for all groups) and had statistically similar intracellular perforin and granzyme-B expression. In vitro CTL migration to a human mesenchymal stem cell line was greatest with IL-15 CTL (30.5%, n=4), followed by IL-7 CTL (18.9%, n=4), and least in IL-2 CTL (17.9%, n=4), though the differences were not significant. In vitro CTL migration was analyzed to an SDF-1α gradient as CXCR4/SDF-1α interactions are necessary for hematopoietic progenitor cell homing to the bone marrow. IL-15 cultured CTL showed the highest migration (48.8%, n=8) as compared to IL-2 (21.7%, n=6, p=0.048) or IL-7 CTL (35.9%, n=8, p>0.05). However, surface expression of CXCR4 measured by flow cytometry was significantly higher in IL-7 CTL (89.4%, n=9) compared to IL-2 CTL (52.2%, n=9, p<0.001) and IL-15 CTL (65.4%, n=10, p=0.002). Experiments are currently underway to further evaluate the role of CXCR4/SDF-1α in GVL. Preliminary in vivo experiments do not suggest any significant differences in CTL engraftment when evaluated at 24 hours post injection. Expression of the anti-apoptotic bcl-2 protein was greatest on IL-7 (MFI=5295, n=13) and IL-15 (MFI=4865, n=14) when compared to IL-2 CTL (MFI=3530, n=13, p=0.02 vs. IL-7, p=0.05 vs. IL-15), suggesting an increased in vivo survival ability. We hypothesize that IL-15 cultured CTL have greater GVL effects due to either higher in vivo survival, greater bone marrow homing efficiency, or both. Future experiments are planned to evaluate in vivo administration of IL-2 to enhance CTL survival in the host. In conclusion, IL-15 cultured CTL had significantly greater in vivo GVL effects compared to IL-2 and IL-7 CTL in the NOD/scid mouse model. This model can be utilized to evaluate the mechanism of T cell mediated GVL against ALL and potentially other human malignancies.

scholarly journals Role of Surfactant Proteins A, D, and C1q in the Clearance of Apoptotic Cells In Vivo and In Vitro: Calreticulin and CD91 as a Common Collectin Receptor Complex

2002 ◽  
Vol 169 (7) ◽  
pp. 3978-3986 ◽  
Author(s):  
R. William Vandivier ◽  
Carol Anne Ogden ◽  
Valerie A. Fadok ◽  
Peter R. Hoffmann ◽  
Kevin K. Brown ◽  
...  
Keyword(s):  
Surfactant Proteins ◽  
Apoptotic Cells ◽  
Receptor Complex

scholarly journals Overexpression of membrane metalloendopeptidase inhibits substance P stimulation of cholangiocarcinoma growth

2014 ◽  
Vol 306 (9) ◽  
pp. G759-G768 ◽  
Author(s):  
Fanyin Meng ◽  
Sharon DeMorrow ◽  
Julie Venter ◽  
Gabriel Frampton ◽  
Yuyan Han ◽  
...  
Keyword(s):  
Substance P ◽  
Functional Role ◽  
Xenograft Model ◽  
Subsequent Increase ◽  
Proliferation In Vitro ◽  
Proteolytic Product ◽  
Stimulation Of

Substance P (SP) promotes cholangiocyte growth during cholestasis by activating its receptor, NK1R. SP is a proteolytic product of tachykinin (Tac1) and is deactivated by membrane metalloendopeptidase (MME). This study aimed to evaluate the functional role of SP in the regulation of cholangiocarcinoma (CCA) growth. NK1R, Tac1, and MME expression and SP secretion were assessed in human CCA cells and nonmalignant cholangiocytes. The proliferative effects of SP (in the absence/presence of the NK1R inhibitor, L-733,060) and of L-733,060 were evaluated. In vivo, the effect of L-733,060 treatment or MME overexpression on tumor growth was evaluated by using a xenograft model of CCA in nu/nu nude mice. The expression of Tac1, MME, NK1R, PCNA, CK-19, and VEGF-A was analyzed in the resulting tumors. Human CCA cell lines had increased expression of Tac1 and NK1R, along with reduced levels of MME compared with nonmalignant cholangiocytes, resulting in a subsequent increase in SP secretion. SP treatment increased CCA cell proliferation in vitro, which was blocked by L-733,060. Treatment with L-733,060 alone inhibited CCA proliferation in vitro and in vivo. Xenograft tumors derived from MME-overexpressed human Mz-ChA-1 CCA cells had a slower growth rate than those derived from control cells. Expression of PCNA, CK-19, and VEGF-A decreased, whereas MME expression increased in the xenograft tumors treated with L-733,060 or MME-overexpressed xenograft tumors compared with controls. The study suggests that SP secreted by CCA promotes CCA growth via autocrine pathway. Blockade of SP secretion and NK1R signaling may be important for the management of CCA.

scholarly journals Ventral pallidal GABAergic neurons control wakefulness associated with motivation through the ventral tegmental pathway

2020 ◽  
Author(s):  
Ya-Dong Li ◽  
Yan-Jia Luo ◽  
Wei Xu ◽  
Jing Ge ◽  
Yoan Cherasse ◽  
...  
Keyword(s):  
Dopaminergic Neurons ◽  
Gabaergic Neurons ◽  
Population Activity ◽  
Lateral Habenula ◽  
Optogenetic Stimulation ◽  
Ventral Tegmental ◽  
Stimulation Of

Abstract The ventral pallidum (VP) regulates motivation, drug addiction, and several behaviors that rely on heightened arousal. However, the role and underlying neural circuits of the VP in the control of wakefulness remain poorly understood. In the present study, we sought to elucidate the specific role of VP GABAergic neurons in controlling sleep–wake behaviors in mice. Fiber photometry revealed that the population activity of VP GABAergic neurons was increased during physiological transitions from non-rapid eye movement (non-REM, NREM) sleep to either wakefulness or REM sleep. Moreover, chemogenetic and optogenetic manipulations were leveraged to investigate a potential causal role of VP GABAergic neurons in initiating and/or maintaining arousal. In vivo optogenetic stimulation of VP GABAergic neurons innervating the ventral tegmental area (VTA) strongly promoted arousal via disinhibition of VTA dopaminergic neurons. Functional in vitro mapping revealed that VP GABAergic neurons, in principle, inhibited VTA GABAergic neurons but also inhibited VTA dopaminergic neurons. In addition, optogenetic stimulation of terminals of VP GABAergic neurons revealed that they promoted arousal by innervating the lateral hypothalamus, but not the mediodorsal thalamus or lateral habenula. The increased wakefulness chemogenetically evoked by VP GABAergic neuronal activation was completely abolished by pretreatment with dopaminergic D1 and D2/D3 receptor antagonists. Furthermore, activation of VP GABAergic neurons increased exploration time in both the open-field and light–dark box tests but did not modulate depression-like behaviors or food intake. Finally, chemogenetic inhibition of VP GABAergic neurons decreased arousal. Taken together, our findings indicate that VP GABAergic neurons are essential for arousal related to motivation.

Influence of mechanical stretch on growth and protein turnover of rat uterus

1988 ◽  
Vol 254 (5) ◽  
pp. E543-E548 ◽  
Author(s):  
A. J. Douglas ◽  
E. W. Clarke ◽  
D. F. Goldspink
Keyword(s):  
Mechanical Stretch ◽  
Rat Uterus ◽  
Dna Contents ◽  
Extensive Growth ◽  
A New Technique ◽  
Rna And Dna ◽  
Stimulation Of

A new technique has been developed and used to distend the uterus of nonpregnant rats for up to 5 days. Continuous distension of the saline-filled uterus induced rapid and extensive growth of the whole uterus and the myometrium by a combination of hyperplasia and hypertrophy. In both cases, 1 day after this imposition of mechanical stretch significant increases (25-50%) in the protein, RNA, and DNA contents were found, with larger changes (100-250%) being progressively expressed up to 5 days. This stretch-induced growth primarily results from a stimulation of protein synthesis (measured both in vivo and in vitro), with little or no change being evident in the rate of protein breakdown. These findings have been discussed in relation to the role of stretch in the growth of the uterus during pregnancy and stretch-induced responses found in other types of muscle.

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