Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Flow cytometry (FC) provides a reproducible investigation of cell surface antigens on platelets. The aim of this study was to elaborate appropriate protocols and to compare them with other techniques that have already been published. (1) Venipuncture with tubes containing citrate was better for the preservation of the antigenicity than using ACD tubes. The isolated platelets could not be completely distinguished from detritus and protein aggregates. Therefore a platelet concentration between 10(7) and 10(8)/ml measurement buffer was necessary to obtain a sufficient resolution by FC. (2) Isolation methods using either differential centrifugation or diluted Ficoll-Hypaque as a flotation medium provided platelets of equal purity. The method with Ficoll-Hypaque resulted in a higher number of isolated platelets than differential centrifugation. The demonstration of platelets and their antigens in whole blood without isolation gave good results provided the platelets were not activated. Activation of platelets with 1 NIH-U thrombin/l resulted in the loss of a part of the highly activated platelets because of their aggregation. (3) Comparing different concentrations of paraformaldehyde in PBS, fixation with 1% for 15 min provided the best antigen preservation for most of the antigens investigated. Isolation induced platelet activation. In order to avoid this effect, the whole anticoagulated blood was fixed with 1% paraformaldehyde for 15 min immediately after venipuncture. Then the platelets were isolated using diluted Ficoll-Hypaque. In this way, systemic activation of platelets can be detected with antibodies against glycoproteins which are translocated from the alpha-granules or lysosomes to the cell membrane. These activation markers can be determined on immediately fixed platelets (already in the whole blood) without any interference due to unspecific activation caused by the isolation procedure. (4) Platelet treatment with citric acid at pH 3, in order to remove the antigenicity of HLA-class I molecules, was sensitive to immediate fixation with paraformaldehyde in the whole blood. Fixation after isolating the platelets made it possible to demonstrate antigen stripping, and the free heavy chain, devoid of the beta 2-microglobulin, could be clearly demonstrated. (5) Using standardization beads, the average number of antigenic sites per platelet could be determined for the investigated specificities. It was shown that antibodies which have been directly conjugated or biotinylated and combined with streptavidin-phycoerythrin yielded similar results in terms of the number of antigenic binding sites while unconjugated antibodies in combination with FITC-conjugated anti-mouse-IgG led to overestimation of antigenic binding sites.
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PMID:Standardization of the flow cytometric determination of HLA class I antigens, 'platelet-specific' glycoproteins and activation markers. 776 17

In this paper, we report the purification and partial characterization of human platelet aggregation factor form the extracellular products (ECP) of Streptococcus mitis (S. mitis) isolated from a patient with Kawasaki disease (KD). Platelet aggregation reaction was carried out using platelet-rich plasma (PRP) and washed platelets suspended in ACD-PBS. The aggregation factor was designated as S. mitis-derived human platelet aggregation factor (Sm-hPAF). The results obtained were as follows. 1) Sm-hPAF was isolated by chromatography on DEAE-Sepharose CL-6 B, hydroxyapatite and Superdex 75 columns. The purified Sm-hPAF showed a single band upon SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular weight of approximately 66 kDa on SDS-PAGE. The isoelectric point (pI) of Sm-hPAF was 8.5, and Sm-hPAF showed an absorption peak at 278 nm on absorption spectra. When the platelet aggregation activity of the Sm-hPAF was compared with that of ECP, the specific aggregation activity of the of Sm-hPAF was significantly increased (up to 28-fold). Sugars were not found in Sm-hPAF. The sequence of the first 15 amino-terminal amino acid residues were H.Asp-Glu-Gln-Gly-Asn-Arg-Pro-Val-Glu-Thr-Glu-Asn-Ile-Ala-Arg. The platelet aggregation activity of Sm-hPAF was inactivated by heating at 45 degrees C for 10 min. 2) PGE2 was released from platelets after incubation for 10 min with Sm-hPAF in a dose-dependent fashion. Platelet aggregation by the Sm-hPAF was totally inhibited by either PGE1, or GRGDS, but these reagents did not inhibit the platelet aggregation by collagen. 3) Histological examination of the rabbit skin sites showing an early reaction revealed increased dilatation of the veins and capillaries with cellular infiltration in the perivascular space of the dermis. Hyperplasia of the endothelial cells was noted. Degeneration of the vascular walls was observed in the later stages of the reaction. Aggregation of red cells in the vascular endothelium was also observed. Sm-hPAF was capable of producing vasculitis. 4) Twenty (76.9%) platelet-rich plasma samples (PRP) derived from 26 healthy human volunteers reacted with Sm-hPAF, but the remaining 6 PRPs were not reactive. Preliminary study suggests the existence of an inhibitory factor in plasma from nonreactive donors.
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PMID:[Purification and partial characterization of a novel human platelet aggregation factor in the extracellular products of Streptococcus mitis, strain Nm-65]. 898 63

Haematopoietic stem cell transplantation is indicated in several haematologic and genetic diseases, the most notable being aplastic anemia and leukemias. Bone marrow has been the traditional source of these cells. Human umbilical cord blood (UCB) has recently become an alternative source of haematopoietic stem cells for transplants. The advantages of cord blood include noninvasive collection without risk to mother and neonate, low risk of viral infection, and immunologic immaturity of cord cells. Single umbilical cord blood donation is usually sufficient for transplantation to adult recipients. Additionally, banking of HLA-typed UCB appears valuable in patients lacking a family donor. This study has focused on basic "perinatological" parameters of umbilical cord blood: average volume of single donation UCB and initial storage conditions before isolation of haematopoietic stem cells. Additionally, the mean content of CD34+ haematopoietic stem cells in leukocyte, lymphocyte and mononuclear cell fractions was established. Correlations between levels of so-called pro-inflammatory cytokines (present in cord blood serum) and number, viability and clonogenicity of cord blood mononuclear cells were checked. UCB samples were obtained by "open" collection during vaginal deliveries and cesarean sections. The collected blood was stored in solutions of anticoagulants (ACD, CPDA-1, heparin) and culture media (PBS, Iscove medium, RPMI), during several time intervals (0-1 h, 1-6 h, 6-12 h, 12-24 h) and at two temperatures (+4 degrees C, ambient). UCB volumes, as well as MNC counts, correlated with delivery type, placental weight, neonatal body weight and duration of pregnancy. The concentration, viability and clonogenicity of MNCs were assessed after collection and storage. The subpopulation of CD34+ haematopoietic stem cells was isolated from MNCs using monoclonal antibodies and magnetic-based separation. The number, viability and clonogenicity of CD34+ cells were evaluated. Subsequently in some samples, the concentration of proinflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8, and TNF-alpha), number of mononuclear cells and in vitro clonogenicity of myeloid progenitors (CFU-GM) were determined. It was found that the collected blood volume depended on neonatal body weight (Fig. 1). Umbilical blood could be stored either at ambient temperature (Fig. 4) or +4 degrees C (recommended because of reduced risk of infection) for up to 24 hours in RPMI solution (Fig. 5) with heparin (Fig. 2, 3). CD34+ cell count correlated with mononuclear cell count only (Fig. 6). A negative correlation between the number of mononuclear cells and concentration of TNF-alpha was revealed (Fig. 7), as well as between the number of detectable CFU-GM and concentration of IL-1 beta (Fig. 8). In conclusion, UCB collection and short-term storage is a safe and simple method for graftable haematopoietic stem cell recovery. Save for IL-1 beta and TNF-alpha, cytokine levels did not correlate with the studied parameters of umbilical cord blood.
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PMID:[Improved method for delivery room collection and storage of human cord blood cells for grafting]. 1251 5