scholarly journals Senescence of canine biotinylated erythrocytes: increased autologous immunoglobulin binding occurs on erythrocytes aged in vivo for 104 to 110 days

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3469-3473 ◽  
Author(s):  
JA Christian ◽  
AH Rebar ◽  
GD Boon ◽  
PS Low
Keyword(s):  
Life Span ◽  
Cell Sorting ◽  
Gradient Centrifugation ◽  
Magnetic Cell Sorting ◽  
Autologous Plasma ◽  
Low Degree ◽  
La Jolla ◽  
Immunoglobulin Binding

Abstract We have evaluated senescence related changes in canine red blood cells (RBCs) using the biotinylation system, where RBCs are labeled in vivo with biotin at the beginning of their life span, and retrieved from circulation on immobilized avidin at the end of their life span. This approach avoids the controversial use of density gradient centrifugation to collect presumably old RBCs. Furthermore, the dog is an appropriate model for human RBC senescence because it has a low degree of random RBC loss and a similarly long RBC life span (approximately 110 days). Two dogs had 97% to 100% of their circulating RBCs biotinylated by infusion of N-hydroxysuccinimido biotin (Clontech, Palo Alto, CA; Calbiochem, La Jolla, CA) dissolved in dimethyl sulfoxide. At postbiotinylation days 104 and 107 for one dog and day 110 for the other dog, biotinylated RBCs were isolated by magnetic cell sorting and analyzed for the presence of autologous IgG using 125I- labeled sheep-antidog IgG (SAD IgG). On all 3 days, there were at least three times more SAD IgG molecules per RBC on senescent biotinylated RBCs than on control (unfractionated) RBCs (day 104: 11,677 v 3,399; day 107: 6,710 v 2,115; day 110: 6,042 v 1,838 molecules of SAD IgG per senescent v control RBC). Furthermore, it is unlikely that an immune response to the conjugated biotin had been elicited, because fresh in vitro biotinylated RBCs that were incubated in autologous plasma (taken after exposure to circulating biotinylated RBCs for 113 days) and then exposed to the SAD IgG showed no increase in antibody binding over control (non-biotinylated) RBCs (1,431 v 1,378 cpm/10(8) biotinylated v control RBCs; P > .20). These results suggest that senescence of canine biotinylated RBCs is characterized by binding of autologous IgG and that antibiotin antibodies do not contribute to this process.

scholarly journals Senescence of canine biotinylated erythrocytes: increased autologous immunoglobulin binding occurs on erythrocytes aged in vivo for 104 to 110 days

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3469-3473 ◽  
Author(s):  
JA Christian ◽  
AH Rebar ◽  
GD Boon ◽  
PS Low
Keyword(s):  
Life Span ◽  
Cell Sorting ◽  
Gradient Centrifugation ◽  
Magnetic Cell Sorting ◽  
Autologous Plasma ◽  
Low Degree ◽  
La Jolla ◽  
Immunoglobulin Binding

We have evaluated senescence related changes in canine red blood cells (RBCs) using the biotinylation system, where RBCs are labeled in vivo with biotin at the beginning of their life span, and retrieved from circulation on immobilized avidin at the end of their life span. This approach avoids the controversial use of density gradient centrifugation to collect presumably old RBCs. Furthermore, the dog is an appropriate model for human RBC senescence because it has a low degree of random RBC loss and a similarly long RBC life span (approximately 110 days). Two dogs had 97% to 100% of their circulating RBCs biotinylated by infusion of N-hydroxysuccinimido biotin (Clontech, Palo Alto, CA; Calbiochem, La Jolla, CA) dissolved in dimethyl sulfoxide. At postbiotinylation days 104 and 107 for one dog and day 110 for the other dog, biotinylated RBCs were isolated by magnetic cell sorting and analyzed for the presence of autologous IgG using 125I- labeled sheep-antidog IgG (SAD IgG). On all 3 days, there were at least three times more SAD IgG molecules per RBC on senescent biotinylated RBCs than on control (unfractionated) RBCs (day 104: 11,677 v 3,399; day 107: 6,710 v 2,115; day 110: 6,042 v 1,838 molecules of SAD IgG per senescent v control RBC). Furthermore, it is unlikely that an immune response to the conjugated biotin had been elicited, because fresh in vitro biotinylated RBCs that were incubated in autologous plasma (taken after exposure to circulating biotinylated RBCs for 113 days) and then exposed to the SAD IgG showed no increase in antibody binding over control (non-biotinylated) RBCs (1,431 v 1,378 cpm/10(8) biotinylated v control RBCs; P > .20). These results suggest that senescence of canine biotinylated RBCs is characterized by binding of autologous IgG and that antibiotin antibodies do not contribute to this process.

scholarly journals Sickling-induced binding of immunoglobulin to sickle erythrocytes

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 636-639 ◽  
Author(s):  
GA Green ◽  
VK Kalra
Keyword(s):  
Flow Cytometry ◽  
Gradient Centrifugation ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Low Density ◽  
Autologous Plasma ◽  
Sickle Cells ◽  
Sickle Erythrocytes

Abstract Previously we demonstrated that sickle erythrocytes sedimenting at high densities after gradient centrifugation contain higher levels of surface immunoglobulin bound in vivo in comparison to low-density erythrocytes from the same patient. The present study examines the possibility that binding of autologous IgG to sickle erythrocytes may be associated with the sickling phenomenon. In the present study we subjected low-density erythrocytes to prolonged sickling under nitrogen in the presence of platelet-poor autologous plasma with added glucose for 24 hours (37 degrees C). After reoxygenation IgG bound in vitro was quantified by a nonequilibrium 125iodinated protein A-binding assay and by flow cytometry. Results show that sickle erythrocytes incubated under nitrogen bound significantly (P less than .001) more IgG, 439 +/- 41, molecules of IgG per cell (mean +/- SD) compared with sickle cells incubated under oxygenation (227 +/- 12 molecules of IgG per red cell) or compared with 196 +/- 26 molecules IgG per cell for untreated sickle cells. In contrast, normal erythrocytes incubated in autologous plasma exhibited no detectable IgG binding in vitro under either oxygenation or deoxygenation. Flow cytometry shows that deoxygenation of sickle cells generated a two-to-sixfold increase in the subpopulation of brightly fluorescent IgG-positive cells in comparison to oxygenated sickle cells and a 13.5% +/- 3.1% (mean +/- SD) increase in median fluorescence intensity for fluorescein isothiocyanate-labeled deoxygenated sickled cells compared with labeled oxygenated sickle cells. Our studies demonstrate that prolonged sickling will induce in vitro binding of autologous IgG to sickle erythrocytes. These findings indicate that sickle erythrocytes may be unique when compared with erythrocytes from other nonimmunologic hemolytic anemias or senescent red cells in that the primary events producing surface antigens recognized by autoantibody may include the sickling process. These findings also suggest that sickling in vivo may generate membrane alterations in sickle erythrocytes that lead to cumulative binding of autoantibody in vivo.

scholarly journals Magnetic Cell Sorting for In Vivo and In Vitro Astrocyte, Neuron, and Microglia Analysis

2019 ◽  
Vol 88 (1) ◽  
Author(s):  
Leanne M. Holt ◽  
S. Tristan Stoyanof ◽  
Michelle L. Olsen
Keyword(s):  
Cell Sorting ◽  
Magnetic Cell Sorting

scholarly journals Sickling-induced binding of immunoglobulin to sickle erythrocytes

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 636-639
Author(s):  
GA Green ◽  
VK Kalra
Keyword(s):  
Flow Cytometry ◽  
Gradient Centrifugation ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Low Density ◽  
Autologous Plasma ◽  
Sickle Cells ◽  
Sickle Erythrocytes

Previously we demonstrated that sickle erythrocytes sedimenting at high densities after gradient centrifugation contain higher levels of surface immunoglobulin bound in vivo in comparison to low-density erythrocytes from the same patient. The present study examines the possibility that binding of autologous IgG to sickle erythrocytes may be associated with the sickling phenomenon. In the present study we subjected low-density erythrocytes to prolonged sickling under nitrogen in the presence of platelet-poor autologous plasma with added glucose for 24 hours (37 degrees C). After reoxygenation IgG bound in vitro was quantified by a nonequilibrium 125iodinated protein A-binding assay and by flow cytometry. Results show that sickle erythrocytes incubated under nitrogen bound significantly (P less than .001) more IgG, 439 +/- 41, molecules of IgG per cell (mean +/- SD) compared with sickle cells incubated under oxygenation (227 +/- 12 molecules of IgG per red cell) or compared with 196 +/- 26 molecules IgG per cell for untreated sickle cells. In contrast, normal erythrocytes incubated in autologous plasma exhibited no detectable IgG binding in vitro under either oxygenation or deoxygenation. Flow cytometry shows that deoxygenation of sickle cells generated a two-to-sixfold increase in the subpopulation of brightly fluorescent IgG-positive cells in comparison to oxygenated sickle cells and a 13.5% +/- 3.1% (mean +/- SD) increase in median fluorescence intensity for fluorescein isothiocyanate-labeled deoxygenated sickled cells compared with labeled oxygenated sickle cells. Our studies demonstrate that prolonged sickling will induce in vitro binding of autologous IgG to sickle erythrocytes. These findings indicate that sickle erythrocytes may be unique when compared with erythrocytes from other nonimmunologic hemolytic anemias or senescent red cells in that the primary events producing surface antigens recognized by autoantibody may include the sickling process. These findings also suggest that sickling in vivo may generate membrane alterations in sickle erythrocytes that lead to cumulative binding of autoantibody in vivo.

Influence of Platelet Membrane Sialic Acid and Platelet-Associated IgG on Ageing and Sequestration of Blood Platelets in Baboons

1993 ◽  
Vol 70 (04) ◽  
pp. 676-680 ◽  
Author(s):  
H F Kotzé ◽  
V van Wyk ◽  
P N Badenhorst ◽  
A du P Heyns ◽  
J P Roodt ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Life Span ◽  
Blood Platelets ◽  
Senescent Cell ◽  
Platelet Membrane ◽  
Antigen Complex ◽  
The Mean ◽  
Aggregation Of Platelets

SummaryPlatelets were isolated from blood of baboons and treated with neuraminidase to remove platelet membrane sialic acid, a process which artificially ages the platelets. The platelets were then labelled with 111In and their mean life span, in vivo distribution and sites of Sequestration were measured. The effect of removal of sialic acid on the attachment of immunoglobulin to platelets were investigated and related to the Sequestration of the platelets by the spleen, liver, and bone marrow. Removal of sialic acid by neuraminidase did not affect the aggregation of platelets by agonists in vitro, nor their sites of Sequestration. The removal of 0.51 (median, range 0.01 to 2.10) nmol sialic acid/108 platelets shortened their life span by 75 h (median, range 0 to 132) h (n = 19, p <0.001), and there was an exponential correlation between the shortening of the mean platelet life span and the amount of sialic acid removed. The increase in platelet-associated IgG was 0.112 (median, range 0.007 to 0.309) fg/platelet (n = 25, p <0.001) after 0.79 (median, range 0.00 to 6.70) nmol sialic acid/108 platelets was removed (p <0.001). There was an exponential correlation between the shortening of mean platelet life span after the removal of sialic acid and the increase in platelet-associated IgG. The results suggest that platelet membrane sialic acid influences ageing of circulating platelets, and that the loss of sialic acid may have exposed a senescent cell antigen that binds IgG on the platelet membrane. The antibody-antigen complex may then provide a signal to the macrophages that the platelet is old, and can be phagocytosed and destroyed.

scholarly journals Tumor-Targeting Peptides Search Strategy for the Delivery of Therapeutic and Diagnostic Molecules to Tumor Cells

2020 ◽  
Vol 22 (1) ◽  
pp. 314
Author(s):  
Maria D. Dmitrieva ◽  
Anna A. Voitova ◽  
Maya A. Dymova ◽  
Vladimir A. Richter ◽  
Elena V. Kuligina
Keyword(s):  
Phage Display ◽  
Cell Sorting ◽  
Targeted Delivery ◽  
Tumor Targeting ◽  
Search Strategy ◽  
Tumor Therapy ◽  
Therapeutic Agents ◽  
Phage Libraries

Background: The combination of the unique properties of cancer cells makes it possible to find specific ligands that interact directly with the tumor, and to conduct targeted tumor therapy. Phage display is one of the most common methods for searching for specific ligands. Bacteriophages display peptides, and the peptides themselves can be used as targeting molecules for the delivery of diagnostic and therapeutic agents. Phage display can be performed both in vitro and in vivo. Moreover, it is possible to carry out the phage display on cells pre-enriched for a certain tumor marker, for example, CD44 and CD133. Methods: For this work we used several methods, such as phage display, sequencing, cell sorting, immunocytochemistry, phage titration. Results: We performed phage display using different screening systems (in vitro and in vivo), different phage libraries (Ph.D-7, Ph.D-12, Ph.D-C7C) on CD44+/CD133+ and without enrichment U-87 MG cells. The binding efficiency of bacteriophages displayed tumor-targeting peptides on U-87 MG cells was compared in vitro. We also conducted a comparative analysis in vivo of the specificity of the accumulation of selected bacteriophages in the tumor and in the control organs (liver, brain, kidney and lungs). Conclusions: The screening in vivo of linear phage peptide libraries for glioblastoma was the most effective strategy for obtaining tumor-targeting peptides providing targeted delivery of diagnostic and therapeutic agents to glioblastoma.

The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

scholarly journals Protective effects of saffron and its constituent crocetin to motor symptoms, short life span and rough-eyed phenotypes in the fly models of Parkinson’s disease in vivo

2020 ◽  
Author(s):  
Eiji Inoue ◽  
Takahiro Suzuki ◽  
Yasuharu Shimizu ◽  
Keiichi Sudo ◽  
Haruhisa Kawasaki ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Life Span ◽  
Neurodegenerative Disorder ◽  
Neuronal Cell ◽  
Crocus Sativus ◽  
Motor Symptoms ◽  
Protective Effects

AbstractParkinson’s disease (PD) is a common neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the brain. α-Synuclein is an aggregation-prone neural protein that plays a role in the pathogenesis of PD. In our previous paper, we found that saffron; the stigma of Crocus sativus Linné (Iridaceae), and its constituents (crocin and crocetin) suppressed aggregation of α-synuclein and promoted the dissociation of α-synuclein fibrils in vitro. In this study, we investigated the effect of dietary saffron and its constituent, crocetin, in vivo on a fly PD model overexpressing several mutant α-synuclein in a tissue-specific manner. Saffron and crocetin significantly suppressed the decrease of climbing ability in the Drosophila overexpressing A30P (A30P fly PD model) or G51D (G51D fly PD model) mutated α-synuclein in neurons. Saffron and crocetin extended the life span in the G51D fly PD model. Saffron suppressed the rough-eyed phenotype and the dispersion of the size histogram of the ocular long axis in A30P fly PD model in eye. Saffron had a cytoprotective effect on a human neuronal cell line with α-synuclein fibrils. These data showed that saffron and its constituent crocetin have protective effects on the progression of PD disease in animals in vivo and suggest that saffron and crocetin can be used to treat PD.

CYTOTOXICITY, ANTITUMOUR AND ANTICARCINOGENIC ACTIVITY OF CURCUMA LONGA ESSENTIAL OIL

INDIAN DRUGS ◽  
2014 ◽  
Vol 51 (03) ◽  
pp. 28-34
Author(s):  
V.B Liju ◽  
◽  
K Jeena ◽  
R. Kuttan
Keyword(s):  
Essential Oil ◽  
Oral Administration ◽  
Life Span ◽  
Cell Lines ◽  
Curcuma Longa ◽  
Cytochrome P450 Enzyme ◽  
Dalton’S Lymphoma ◽  
Dalton's Lymphoma

In the present study, we have evaluated the antitumour and anticarcinogenic activity of turmeric essential oil in vivo. Turmeric essential oil was found to have significant in vitro cytotoxic activity against Dalton’s lymphoma ascites cells (DLA) and Ehrlich ascites carcinoma (EAC) cancer cell lines. Concentration needed for 50% cytotoxicity (IC50) was 8 μg for DLA cells and 18 μg to EAC cell lines. Oral administration of turmeric essential oil was found to significantly increase the life span (56.25%) of Dalton’s Lymphoma Ascites (DLA) induced ascites tumour bearing mice as well as significantly reduced (P<0.001) the solid tumours. 3-Methyl cholanthrene induced sarcoma development was also delayed and there was significant increase in the life span of mice after oral administration of turmeric essential oil. Moreover, turmeric essential oil significantly (P<0.001) inhibited phenobarbitone induced cytochrome p450 enzyme activity in rats.

scholarly journals Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA

2007 ◽  
Vol 405 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Jørgen de Jonge ◽  
Johanna M. Leenhouts ◽  
Marijke Holtrop ◽  
Pieter Schoen ◽  
Peter Scherrer ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Gradient Centrifugation ◽  
Cationic Lipid ◽  
Sucrose Density Gradient ◽  
Target Cells ◽  
Sucrose Density Gradient Centrifugation ◽  
Viral Membrane ◽  
Viral Envelopes

Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA–virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA–virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA–virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA–virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.

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