Diurnal variations in blood phenylalanine of PKU infants under different feeding regimes.

Unlabelled: In phenylketonuria (PKU) patients, diurnal fluctuations of blood phenylalanine (Phe) are different from healthy individuals. Until now this pattern has been studied in PKU patients over one year of age.

Objective: The aim of this observational study was to investigate diurnal patterns in PKU infants under one year of age receiving both the natural protein and Phe-free formula at the same time or in an alternating feeding scheme.

Antibodies against aquaporin-4 in neuromyelitis optica: distinction between recurrent and monophasic patients.

The detection of antibodies against aquaporin-4 (AQP4) has improved the diagnosis of neuromyelitis optica (NMO). We evaluated a recently established cell-based anti-AQP4 assay in 273 patients with inflammatory CNS demyelination. The assay had a specificity of 99% and a sensitivity of 56% to detect all NMO patients and of 74% to detect the recurrent NMO patients, similar to the initial studies reported.

Adult patients with well-controlled phenylketonuria tolerate incidental additional intake of phenylalanine.

Background/aims: In patients with phenylketonuria (PKU), target ranges of blood phenylalanine (Phe) concentrations have been tightened in order to improve long-term outcomes. We investigated day-to-day and week-to-week variations in blood Phe concentration and the effect of an additional Phe load.

Methods: We performed a longitudinal study in 6 adult PKU patients.

Mannose-binding lectin deficiency facilitates abdominal Candida infections in patients with secondary peritonitis.

Mannose-binding lectin (MBL) deficiency due to variations in the MBL gene is associated with increased susceptibility to infections. In this study, the association between MBL deficiency and the occurrence of abdominal yeast infection (AYI) in peritonitis patients was examined. Eighty-eight patients with secondary peritonitis requiring emergency laparotomy were included.

Variable mannose-binding lectin expression during postoperative acute-phase response.

Background: Low plasma concentrations and genetic polymorphisms of mannan-binding lectin (MBL) have been associated with infectious disease complications during various conditions. The present study examined the nature and expression of MBL deficiency during a surgery-induced acute-phase response.

Methods: Blood was sampled from 20 consecutive patients before and 1, 3, 5, 7, and 10 days and 6 weeks after a uniform abdominal operation (transhiatal esophagectomy).

Substitution of Pichia pastoris-derived recombinant proteins with mannose containing O- and N-linked glycans decreases specificity of diagnostic tests.

Background: Recombinant proteins from Pichia pastoris need to be fully evaluated before used as diagnostic tools.

Objective: The objective of this study was to investigate whether glycosylation by P. pastoris interferes with the specificity of diagnostic tests.

Recombinant human C1-inhibitor produced in Pichia pastoris has the same inhibitory capacity as plasma C1-inhibitor.

Therapeutic application of the serpin C1-inhibitor (C1-Inh) in inflammatory diseases like sepsis, acute myocardial infarction and vascular leakage syndrome seems promising, but large doses may be required. Therefore, a high-yield recombinant expression system for C1-Inh is very interesting. Earlier attempts to produce high levels of C1-Inh resulted in predominantly inactive C1-Inh.

Determinants in the cytoplasmic domain of P-selectin required for sorting to secretory granules.

P-selectin is a granule membrane protein of platelets and endothelial cells that is expressed at the plasma membrane after cell activation. To determine which residues in its cytoplasmic tail are important for sorting to storage granules during biosynthesis, we expressed P-selectin mutants in AtT-20, a murine cell line with secretory granules that contain the hormone corticotropin ('ACTH'). Immunofluorescence microscopy of permeabilized cells revealed that wild-type P-selectin and mutants with alanine substitutions at 14 different positions in the cytoplasmic tail were concentrated in the tips of the cellular processes, which contain the majority of corticotropin granules.

Identification of a homozygous single base pair deletion in the gene coding for the human platelet glycoprotein Ib alpha causing Bernard-Soulier syndrome.

Bernard-Soulier Syndrome (BSS) is a hereditary bleeding disorder which is caused by the absence or the dysfunction of the platelet glycoprotein Ib/IX/V (GP Ib/IX/V) complex, the major receptor for von Willebrand factor (vWf). BSS is characterized by the presence of giant platelets that show a reduced binding of vWf. Although BSS is a well-characterized disease, and many cases have been described in the literature, the molecular genetic basis of this disorder has been studied in only a few patients.

Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src.

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well.

P-selectin mediates Ca(2+)-dependent adhesion of activated platelets to many different types of leukocytes: detection by flow cytometry.

Previous studies have shown that thrombin-activated platelets interact through the P-selectin with neutrophils and monocytes. To identify other types of leukocytes capable of such an interaction, eosinophils, basophils, and lymphocytes were isolated from whole blood. Binding of these cells to activated platelets was examined in a double immunofluorescence assay and the results show that activated platelets not only bind to neutrophils and monocytes, but also to eosinophils, basophils, and subpopulations of T lymphocytes.

In vitro effect of plasmin on human platelet function in plasma. Inhibition of aggregation caused by fibrinogenolysis.

Background: Plasmin has been reported both to activate platelets and to inhibit platelet functions. The latter effect was thought to be caused by proteolysis of the main membrane glycoproteins.

Methods And Results: We found that incubation of citrated human platelet-rich plasma with streptokinase (SK) (300 IU/ml) does not produce any detectable activation but leads to a time-dependent inhibition of ADP-induced aggregation accompanied by substantial fibrinogenolysis.

Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane.

Platelet glycoprotein (GP) V is a Mr 82,000 plasma membrane protein of unknown function that is cleaved by the potent platelet agonist, thrombin, to yield a Mr 69,500 fragment (GPVf1). Platelet GPIb, a disulfide-linked alpha beta heterodimer (Mr 160,000) that forms a noncovalent complex with GPIX (Mr 22,000), functions as the platelet adhesion receptor for surface-bound von Willebrand factor. Association between GPV and GPIb-IX has been suggested by the finding that both proteins are deficient in the Bernard-Soulier syndrome, a bleeding disorder characterized by giant platelets and defective interaction with von Willebrand factor.

Quantification of platelet-bound immunoglobulins of different class and subclass using radiolabelled monoclonal antibodies: assay conditions and clinical application.

Quantification of platelet-bound immunoglobulins (PBIg) with radiolabelled murine monoclonal antibodies (mAbs) has been described only for IgG so far. Here we describe some modifications of this mAb radioimmunoassay (MARIA) and show that by using a panel of radiolabelled specific mAbs it is possible to quantify not only PBIgG but also PBIgG subclasses and PBIgM. Analysis by gel filtration showed that all anti-IgG and anti-IgG-subclass mAbs bound to their respective antigens in a ratio of about 1:1.

Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8.

The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested.

Detection of platelet activation with monoclonal antibodies and flow cytometry. Changes during platelet storage.

To investigate whether changes in platelet condition during platelet storage correlate with an altered expression of platelet membrane proteins, the binding of monoclonal antibodies (MoAbs) to fresh platelets was compared with MoAbs' binding to thrombin-activated platelets and to platelets stored as platelet concentrates. The MoAbs included antibodies against the platelet glycoprotein (GP) IIb/IIIa complex and against two activation-dependent antigens, one of which was a component of the internal platelet alpha-granule membrane (GMP 140) and the other of which was a 53-kD protein derived from platelet lysosomes. The binding of MoAbs to platelets fixed with 1 percent paraformaldehyde was measured by flow cytometry.

Decreased stability and structural heterogeneity of the residual platelet glycoprotein IIb/IIIa complex in a variant of Glanzmann's thrombasthenia.

A patient is described with a disturbance of platelet function comparable to that in Glanzmann's thrombasthenia. Platelet aggregation and binding of fibrinogen to the patient's platelets were defective and thrombin-induced clot retraction was absent. The platelet fibrinogen content was only moderately reduced.

Human vascular endothelial cells express a membrane protein complex immunochemically indistinguishable from the platelet VLA-2 (glycoprotein Ia-IIa) complex.

Endothelial cells express surface molecules that are involved in cell-matrix interaction, including the vitronectin receptor and the fibronectin receptor, both members of a family of cell adhesion receptors (integrins). Here we provide evidence that endothelial cells express a membrane molecule, indistinguishable from the platelet VLA-2 complex, which is a collagen receptor and a member of the integrin family. To identify this endothelial molecule, we have used a monoclonal antibody, CLB-10G11, which recognizes the VLA-2 complex from platelets.

Laminin receptor on platelets is the integrin VLA-6.

Adhesion of platelets to the subendothelial matrix of an injured vessel wall is an essential step in triggering the formation of a haemostatic plug. Fibronectin, collagen and laminin are three major components of the subendothelial matrix which support platelet adhesion. Receptors for fibronectin and collagen have been identified on platelets and are included in the integrin family.

A monoclonal antibody to the human platelet glycoprotein IIb/IIIa complex induces platelet activation.

The platelet glycoprotein (GP) IIb/IIIa complex functions as the receptor for fibrinogen on activated platelets. The effects of two anti-GPIIb/IIIa monoclonal antibodies on platelet function were studied. These antibodies, 6C9 and C17, recognized different epitopes, which were exclusively present on the undissociated GPIIb/IIIa complex.

The platelet alloantigen Zwa or PlA1 is expressed by cultured endothelial cells.

Recently, the synthesis by cultured human endothelial cells of a membrane protein complex immunologically related to platelet glycoprotein (GP) IIb/IIIa complex was demonstrated. Since platelet GP IIIa is known to carry the platelet alloantigen Zwa or PlA1, studies were performed to establish whether this antigen is also expressed on endothelial cells. The present report describes the results of these studies, which provide evidence for the presence of the Zwa or PlA1 antigen on the surface of cultured human endothelial cells.

Detection of circulating human platelet fragments by using monoclonal antibodies against platelet glycoprotein IIb-IIIa complex.

A radioimmunoassay (RIA) for the detection of platelets and platelet fragments was developed. A sandwich of two monoclonal antibodies directed against the platelet-specific glycoprotein complex IIb-IIIa (GP IIb-IIIa) was used in this assay. A discontinuous 7.