Biomedical subjects

F Tablin

Publications and source records attributed to F Tablin.

At least 19 recordsLinked to original sources

PubMed2005

Equine sperm membrane phase behavior: the effects of lipid-based cryoprotectants.

The plasma membrane of sperm can undergo lipid phase separation during freezing, resulting in irreversible damage to the cell. The objective of our study was to examine the membrane phase behavior of equine spermatozoa in the absence and presence of lipid-based cryoprotectants. Biophysical properties of sperm membranes were investigated with Fourier-transform infrared spectroscopy. Compared to fresh untreated sperm, postthaw untreated sperm showed extensive lipid phase separation and rearrangement. In contrast, postthaw sperm that were cryopreserved in egg phosphatidylcholine (egg PC)- or soy phosphatidylcholine (soy PC)-based diluents showed similar lipid phase behavior to that of fresh, untreated sperm. Studies with a deuterium-labeled PC lipid (POPCd-31) suggest that exogenous lipid from the diluents are strongly associated with the sperm membrane, and scanning electron microscopy images of treated sperm show the presence of lipid aggregates on the membrane surface. Thus, the exogenous lipid does not appear to be integrated into the sperm membrane after cryopreservation. When compared to a standard egg-yolk-based diluent (INRA 82), the soy and egg PC media preserved viability and motility equally well in postthaw sperm. A preliminary fertility study determined that sperm cryopreserved in the soy PC-based medium were capable of fertilization at the same rate as sperm frozen in the conventional INRA 82 medium. Our results show that pure lipid-based diluents can prevent membrane damage during cryopreservation and perform as well as a standard egg-yolk-based diluent in preserving sperm viability, motility, and fertility.

Animals↗

PubMed2001

Thrombocytopenia in a mouse model of human granulocytic ehrlichiosis.

Despite its apparently strict granulocytotropism, thrombocytopenia is a consistent hallmark of infection with the agent of human granulocytic ehrlichiosis (HGE), regardless of host species. Laboratory mice are valuable models of HGE agent infection kinetics and immunity, but initial studies of HGE infection in mouse models have failed to demonstrate thrombocytopenia. More thorough analysis of platelet kinetics, however, reveals a consistent and rapid, marked decrease (50% decline by day 2-4 after infection) in circulating platelet number in both C3H/HeN and C57BL/6J mice during infection with the HGE agent. The roles of splenic consumption and immune-mediated destruction were evaluated as potential mechanisms of the thrombocytopenia. Both splenectomized mice and mice with severe combined immunodeficiency (lacking B and T lymphocytes) became similarly thrombocytopenic in response to infection with the HGE agent. This study validates the appropriateness of the mouse as a model of HGE, including its usefulness for the investigation of thrombocytopenia.

Animals↗

PubMed2001

Northern elephant seal platelets: analysis of shape change and response to platelet agonists.

Blood platelets have a vital role in the maintenance of normal mammalian hemostasis. Rapid pressure changes and temperatures lower than 20 degrees C cause activation of human and terrestrial mammal platelets. Elephant seals are routinely subjected to pressures as high as 150 atm, yet do not suffer from the thrombotic effects of platelet activation associated with rapid decompression. We examined the ultrastructure of Northern elephant seal (Mirounga angustirostris) platelets and their functional and morphological response to various platelet agonists. Unstimulated elephant seal platelets are discoid cells, with a microtubule coil, randomly dispersed alpha and dense granules, and glycogen granules. There are well-defined areas of membranous invaginations that indicate the presence of an open canalicular system (OCS). Aggregometry was used to determine the response of elephant seal platelets to various platelet agonists. Dose-dependent curves were generated for thrombin, collagen, and adenosine diphosphate (ADP). Platelet response to thrombin was dose-dependent and was maximal at 2.5 U/ml. Platelets collected in sodium citrate had blunted responses to both ADP and collagen. ADP stimulation caused only reversible, primary activation (shape change) at > or = 5 microM, while platelets did not aggregate in response to any concentration of collagen. Platelets collected in sodium heparin did respond fully to both to ADP and collagen. There was small, reversible shape change in response to ristocetin, but no response to epinephrine. Decreased sensitivity of elephant seal platelets to agonists may be a protective mechanism developed in response to rapid pressure changes and cold temperatures associated with adaptation to an extreme environment.

Adaptation, Physiological↗

PubMed2001

Human platelets loaded with trehalose survive freeze-drying.

Human blood platelets are stored in blood banks for 5 days, after which they are discarded, by federal regulation. This short lifetime has led to a chronic shortage of platelets, a problem that is particularly acute in immunosuppressed patients, such as those with AIDS. We report here that platelets can be preserved by freeze-drying them with trehalose, a sugar found at high concentrations in organisms that naturally survive drying. We suggest that these findings will obviate the storage problem with platelets. Trehalose is rapidly taken up by human platelets at 37 degrees C, with loading efficiencies of 50% or greater. Fluid-phase endocytosis plays an important role in this efficient uptake of trehalose, but other mechanisms may also be involved. Trehalose-loaded platelets were successfully freeze-dried, with excellent recovery of intact platelets. Rehydration from the vapor phase led to a survival rate of 85%. The response of these platelets to the agonists thrombin (1 U/ml), collagen (2 microg/ml), ADP (20 micromM), and ristocetin (1.6 mg/ml) was almost identical to that of fresh, control platelets. Analysis by Fourier transform infrared spectroscopy demonstrated that the membrane and protein components of trehalose-loaded platelets after freeze-drying, prehydration, and rehydration were remarkably similar to those of fresh platelets.

Biological Transport, Active↗

PubMed2001

The trehalose myth revisited: introduction to a symposium on stabilization of cells in the dry state.

This essay is an introduction to a series of papers arising from a symposium on stabilization of cells in the dry state. Nearly all of these investigations have utilized the sugar trehalose as a stabilizing molecule. Over the past two decades a myth has grown up about special properties of trehalose for stabilization of biomaterials. We review many of such uses here and show that under ideal conditions for drying and storage trehalose has few, if any, special properties. However, under suboptimal conditions trehalose has some distinct advantages and thus may remain the preferred excipient. We review the available mechanisms for introducing trehalose into the cytoplasm of living cells as an introduction to the papers that follow.

Animals↗

PubMed2001

Membrane reorganization during chilling: implications for long-term stabilization of platelets.

This essay is a review of the various biophysical and biochemical events that make up the factors responsible for platelet cold-induced activation. It describes the formation of large membrane domains composed of raft aggregates that occur during chilling and storage. It also presents strong evidence that platelet membranes undergo lateral phase separation during prolonged storage in the cold and suggests that raft aggregation and lateral phase separation are key events which must be obviated to stabilize platelets and store them either in the frozen or in the dry state.

Blood Platelets↗

PubMed2001

Evaluation of glycoprotein Ib expression on feline platelets.

OBJECTIVE: To determine whether platelets obtained from cats expressed glycoprotein Ib (GPIb). SAMPLE POPULATION: Platelets obtained from 11 specific-pathogen-free cats. PROCEDURE: Platelets were analyzed by use of immunofluorescence microscopy, flow cytometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and immunoprecipitation. RESULTS: Immunofluorescence microscopy and flow cytometry revealed the protein on the surface of feline platelets. Biochemical studies (western immunoblot analysis and immunoprecipitation) revealed a 140-kd membrane glycoprotein. Additional biochemical studies revealed that feline GPIb was sensitive to proteolysis, because platelet cytoskeletons prepared with low concentrations of a calpain inhibitor (ie, leupeptin; 100 microg/ml) had substantial proteolysis, and there was an association of protein fragments with the actin cytoskeleton. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these results indicate that feline platelets express a 140-kd membrane protein that is recognized by monoclonal antibodies developed against GPIb. Application of standardized ELISA to quantitate glycocalicin, the water-soluble fragment of GPIb, may provide important information on the production of microvesicles, increased platelet turnover, and abnormal proteolysis.

Animals↗

PubMed2000

Effects of temperature on calcium-sensitive fluorescent probes.

The effect of temperature on the binding equilibria of calcium-sensing dyes has been extensively studied, but there are also important temperature-related changes in the photophysics of the dyes that have been largely ignored. We conducted a systematic study of thermal effects on five calcium-sensing dyes under calcium-saturated and calcium-free conditions. Quin-2, chlortetracycline, calcium green dextran, Indo-1, and Fura-2 all show temperature-dependent effects on fluorescence in all or part of the range tested (5-40 degrees C). Specifically, the intensity of the single-wavelength dyes increased at low temperature. The ratiometric dyes, because of variable effects at the two wavelengths, showed, in general, a reduction in the fluorescence ratio as temperature decreased. Changes in viscosity, pH, oxygen quenching, or fluorescence maxima could not fully explain the effects of temperature on fluorescence. The excited-state lifetimes of the dyes were determined, in both the presence and absence of calcium, using multifrequency phase-modulation fluorimetry. In most cases, low temperature led to prolonged fluorescence lifetimes. The increase in lifetimes at reduced temperature is probably largely responsible for the effects of temperature on the physical properties of the calcium-sensing dyes. Clearly, these temperature effects can influence reported calcium concentrations and must therefore be taken into consideration during any investigation involving variable temperatures.

Aminoquinolines↗

PubMed2000

Animal models for studies on cold-induced platelet activation in human beings.

When human platelets are chilled below about 20 degrees C, they spontaneously activate, a phenomenon that limits their storage lifetime. We have previously shown that this activation in chilled human platelets is associated with passage through a lipid phase transition. Because animal models are necessary for Investigating methods for cold storage of platelets, it is essential to determine whether such phase transitions and chilling-induced activation are found in these models. In this study we examined platelets from some commonly used animal models-pigs, rhesus monkeys, mice, dogs, and rabbits. Using Fourier transform infrared spectroscopy (FTIR), we detected the thermotropic membrane phase transition in Intact platelets and assessed the morphologic response of the platelets to chilling. Statistical analysis of both FTIR and shape change show that of the animal models tested, pig platelets are most similar to human platelets. These studies suggest that pigs and pig platelets are the models of choice for the study of cold-induced platelet activation in human beings.

Animals↗

PubMed1999

The internal calcium concentration of human platelets increases during chilling.

Human platelets must be stored at 22 degreesC in blood banks, because of the well-known phenomenon of cold-induced activation. When human platelets are chilled below room temperature, they undergo shape change and vesicle secretion that resembles physiological agonist-mediated activation. The trigger for the cascade of events leading to platelet activation at hypothermic temperatures is not known, although an increase in the internal calcium concentration ([Ca]i) due to passage of the platelet membranes through their thermotropic phase transition has been proposed. We report here that the fluorescent calcium-sensitive probe, Indo-1, has been used to estimate the internal calcium concentration of human platelets during a reduction in temperature from 20 degreesC to 5 degreesC at a rate of 0.5 degreesC/min. An increase on the order of 100 nM was recorded. Almost all of the increase in [Ca2+]i occurs during the chilling process, as incubation of platelets for 1 h at low temperature did not lead to a continued calcium concentration increase. The increase in [Ca2+]i during chilling is likely to be due to more than a single mechanism, but might include some release of the calcium stores from the dense tubule system. Loading platelets with the calcium chelator BAPTA (1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) dramatically reduced the increase in [Ca2+]i seen during chilling. Antifreeze glycoproteins (AFGPs) isolated from the blood serum of Antarctic fishes, which are known to protect platelets from cold-induced activation, did not eliminate the rise in [Ca2+]i during chilling, suggesting that signaling mechanisms are likely to be involved in cold-induced activation.

Antifreeze Proteins↗

PubMed1999

Are lipid phase transitions responsible for chilling damage in human platelets?

In previous studies we have proposed that the well-known chilling-induced activation of human blood platelets can be ascribed at least in part to a thermotropic phase transition in membrane lipids. The evidence that this is the case is reviewed and amplified in this review, followed by an examination of the available physical data concerning phase transitions in lipid mixtures that mimic the mixture found in platelet membranes. Assuming complete mixing at all temperatures and equal contributions of the members of the mixture to the phase transition, the lipid mixture found in platelets should give values for Tm ranging from about 1 degrees C to about 16 degrees C, depending on the isomers present in the mixture. (The former value is not in agreement with the observed Tm, but the latter is in excellent agreement.) However, examination of the phase diagram for a binary pair of lipids found in platelet membranes shows that ideal mixing almost certainly does not occur; instead of a linear phase diagram, a convex one was obtained. This shape for the phase diagram, which would displace Tm to an unexpectedly elevated temperature, is in agreement with previously published phase diagrams for mixtures of this type. The prediction, based on thermodynamic properties of lipids found in the platelets, is that Tm will be displaced upward in more complex mixtures of the composition found in platelets, a prediction that requires experimental testing.

Blood Platelets↗

PubMed1999

Platelet-erythrocyte adhesion in sickle cell disease.

BACKGROUND: The abnormal adherence of sickle red blood cells (sRBC) to other cell types likely contributes to vaso-occlusion. Increased numbers of platelet-erythrocyte aggregates (PEA) and platelet activation have been described in sickle cell disease. The present study was undertaken to determine the contribution, if any, of the extracellular matrix protein thrombospondin to the adhesion of sRBC and platelets. METHODS: Platelet activation and PEA were measured using fluorescent-labeled monoclonal antibodies and flow cytometry. Platelet red-cell adhesion was measured by a gravity sedimentation assay. Erythrocyte-bound thrombospondin (TSP) was determined by enzyme-linked immunoabsorbant assay (ELISA). RESULTS: Our studies demonstrate significant platelet activation and adhesion of sRBC to platelets in sickle cell disease. Thrombospondin was detected on sRBC. There was variable inhibition of sRBC-platelet adhesion by antibodies to CD36 (thrombospondin receptor) and antibodies to thrombospondin. CONCLUSIONS: Thrombospondin on sRBC may mediate, at least in part, sRBC-platelet adhesion in sickle cell disease. The study of heterotypic cell-cell interactions is important in understanding the pathogenesis of vaso-occlusion in sickle cell disease.

Antibodies, Monoclonal↗

PubMed1998

A novel tetraester construct that reduces cationic lipid-associated cytotoxicity. Implications for the onset of cytotoxicity.

The preparation of cationic amphiphiles that induce minor cytotoxic response during polynucleotide delivery into mammalian cells has been limited by the conventional use of ester, amide, or carbamate linkages to tether either the polar or the hydrophobic domains. The deleterious effects of ammonium-based lipidic salts on cellular processes have been well-established. The present report is the first example of a linchpin tetraester construct that utilizes ester linkages to tether both the polar and hydrophobic domains. Dimyristoyl and dioleoyl analogues were prepared from pentaerythritol, N,N-dimethylglycine, and their corresponding fatty acyl groups via successive diesterifications followed by amine quaternization. The resultant cationic tetraesters were examined in transfection (luciferase) and cell proliferation (MTS) assays using NIH 3T3 and 16HBE14o- cells. The tetraesters exhibited transfection activity comparable to the well-studied lipids DOTAP and DC-cholesterol (DC-chol) in both cell lines. The tetraester construct afforded no cytotoxicity in NIH3T3 cells and provided a significant lowering of cytotoxicity relative to DC-chol in the 16HBE14o- cells. The expression of green fluorescent protein (GFP) in both cell lines also was examined using the lipid panel. Comparison of fluorescent and corresponding phase-contrast images confirmed the chemical cytotoxicity results and revealed that the cytotoxic response was not dependent on transgene expression. Phase-contrast micrographs of cells treated with the cationic lipid panel in the absence of GFP plasmid showed identical morphology to the GFP-transfected cells, suggesting that the onset of a lipid-mediated cytotoxic response might occur at a stage prior to endosomal encapsulation.

3T3 Cells↗

PubMed1998

Platelet activation in patients with sickle cell disease.

Vascular occlusion and vasculopathy underlie much of the morbidity in patients with sickle cell anaemia. Platelets may play a role in this vasculopathy. Samples from 43 patients with sickle cell disease (SCD) were examined for evidence of platelet activation using fluorescent-labelled monoclonal antibodies and flow cytometry. There was increased expression of activation-dependent antigens on the platelets from patients with SCD compared to those from both Caucasian and African-American controls. In addition, SCD patients had increased levels of platelet microparticles. Platelets are activated in patients with sickle cell disease. The contribution of platelet activation to sickle cell pathophysiology is under active investigation in our laboratories.

Adolescent↗

PubMed1998

Determination of time-dependent shape changes in red blood cells.

A kinetic method for detecting changes in red blood cell shape with time resolution on the order of seconds is described. The definition of shape index (SI) and the experimental conditions under which the measurements should be conducted are given, and the relationship between SI and morphological index, which is often used to quantitatively assess red blood cell morphology, is established. Further, we show that the method can be used to assess shape changes under a variety of experimental conditions known to cause major perturbations in the morphology of red blood cells (such as addition of lysolecithin or chlorpromazine) and to detect changes in shape during chemical fixation for observation with microscopy. We report here that the ability of glutaraldehyde to fix red blood cells without additional alteration in shape strongly depends on the morphology that is being fixed and the initial state of the cells.

Adult↗

PubMed1997

Effect of recombinant interleukin-6 and thrombopoietin on isolated guinea pig bone marrow megakaryocyte protein phosphorylation and proplatelet formation.

Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100ng/mal IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 microg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAK1 and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation.

1-Methyl-3-isobutylxanthine↗

PubMed1997

Platelet activation and platelet-erythrocyte aggregates in patients with sickle cell anemia.

Vascular occlusion and vasculopathy underlie much of the morbidity in patients with sickle cell anemia. Platelets may play a role in this vasculopathy. Samples from 12 adults patients with sickle cell anemia were examined for evidence of platelet activation and formation of platelet-erythrocyte aggregates (PEA) using fluorescent-labeled monoclonal antibodies and flow cytometry. We noted an increased expression of activation-dependent antigens on the platelets from patients with sickle cell anemia compared with those from both white and black control subjects. In addition, patients with sickle cell anemia had increased levels of platelet microparticles and PEA. Platelets are activated in patients with sickle cell anemia and they adhere to sickle erythrocytes. The significance of this activation and adherence are the subject of further investigation.

Adolescent↗

PubMed1997

Pasteurella multocida enters polarized epithelial cells by interacting with host F-actin.

We investigated the interaction of an avian strain of Pasteurella multocida with the cytoskeleton of MDCK cells, which formed a polarized epithelium when grown on type I collagen coated filters. Bacteria were incubated with MDCK cells for 30 min. 2, 4 and 6 hours and their location and association with the cell cytoskeleton determined by double-label immunofluorescence confocal microscopy. Cells were stained with a polyclonal antiserum to the outer-membrane proteins of P. multocida and with rhodamine phalloidin which specifically binds filamentous (F) actin. Confocal microscopy revealed that bacteria entered the cells by 30 min, and that by 6 hours there was a marked alteration in the actin cytoskeleton in which long filaments were reorganized to discrete foci of short actin filaments, within which were one or more bacteria. Electron microscopy demonstrated that by 2 hours, each bacterium was associated with many short 5-6 nm filaments. Treatment of MDCK cells with cytochalasin D for either 30 minutes or 24 hours prior to infection disrupted the actin cytoskeleton and inhibited entry of P. multocida.

Actin Cytoskeleton↗