In vivo inhibition of low density lipoprotein receptor-related protein improves survival of factor VIII in the absence of von Willebrand factor.
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Animals↗
Biomedical subjects
B R Binder
Publications and source records attributed to B R Binder.
Autoantibody reactivity in a case of Schnitzler's syndrome: evidence for a Th1-like response and detection of IgG2 anti-FcepsilonRIalpha antibodies.
Schnitzler's syndrome is a rare disease characterized by chronic urticaria, monoclonal IgM, and clinical and laboratory signs of inflammation. In a subset of patients, the urticarial lesions cause pruritus. However, the pathophysiology of the disease and the biochemical basis of urticaria are not known. We describe a female patient with Schnitzler's syndrome suffering from chronic urticaria associated with pruritus. The patient's serum was found to contain IgG antibodies recognizing cellular components of the microvasculature. In particular, IgG3 antibodies directed against proteins (14-100 kD) expressed in cultured dermal microvascular endothelial cells and mast cells, were found by immunoblotting. Moreover, IgG2 antibodies specific for the alpha-chain of the FcepsilonRI were detectable. However, the autoantibodies did not mediate histamine release in mast cells or basophils. In patients with IgM paraproteinemia who did not have Schnitzler's syndrome, antibodies against endothelial/mast cells or FcepsilonRI were not detectable. In summary, we describe subclass-specific IgG reactivity against microvascular endothelial cells and mast cells indicating Th1 autoimmunity in a patient with Schnitzler's syndrome. Whether such autoantibodies are recurrently produced in patients with Schnitzler's syndrome and play a role in the pathophysiology of the disease remains to be determined.
Adult↗
Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility.
Protein C inhibitor (PCI) is a nonspecific, heparin-binding serpin (serine protease inhibitor) that inactivates many plasmatic and extravascular serine proteases by forming stable 1:1 complexes. Proteases inhibited by PCI include the anticoagulant activated protein C, the plasminogen activator urokinase, and the sperm protease acrosin. In humans PCI circulates as a plasma protein but is also present at high concentrations in organs of the male reproductive tract. The biological role of PCI has not been defined so far. However, the colocalization of high concentrations of PCI together with several of its target proteases in the male reproductive tract suggests a role of PCI in reproduction. We generated mice lacking PCI by homologous recombination. Here we show that PCI(-/-) mice are apparently healthy but that males of this genotype are infertile. Infertility was apparently caused by abnormal spermatogenesis due to destruction of the Sertoli cell barrier, perhaps due to unopposed proteolytic activity. The resulting sperm are malformed and are morphologically similar to abnormal sperm seen in some cases of human male infertility. This animal model might therefore be useful for analyzing the molecular bases of these human conditions.
Animals↗
Smooth muscle cell migration promoting activity of plasma predicts restenosis in patients with peripheral arterial occlusive disease undergoing angioplasty.
BACKGROUND: Efficacy of percutaneous transluminal angioplasty (PTA) is limited by restenosis occurring in a large proportion of patients. Smooth muscle cell (SMC) migration is a major pathomechanism of restenosis. We studied SMC migration inducing activity of plasma from patients with peripheral arterial occlusive disease (PAOD) undergoing PTA. METHODS AND RESULTS: SMC migration was determined in a two-dimensional assay system after addition of 1/25 plasma dilutions. Mean increase in migration area was 65.5 +/- 33.8% in normal controls and 67.7 +/- 53.2% in patients with PAOD. 6 hours after PTA, plasmatic migration inducing activity was largely unchanged. In 19/30 patients with restenosis (6 months after PTA) migration promoting activity (82.7 +/- 60.0) was significantly higher than in 11/30 patients with patent vessels (41.8 +/- 21.0; p = 0.03). No correlation of clinical risk factors with outcome was observed. A weak correlation was found between plasmatic migration promoting activity and levels of epidermal growth factor and transforming growth factor-beta. CONCLUSION: The capacity of human plasma to stimulate SMC migration in tissue culture can be used to assess the risk for restenosis after PTA in patients with PAOD.
Aged↗
Synthesis and ultrastructural localization of protein C inhibitor in human platelets and megakaryocytes.
The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/2 x 10(9) cells. Its specific activity was 5 times higher than that of plasma PCI. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (M(r)) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in alpha granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization.
Blood Platelets↗
Vascular endothelial cell growth factor-induced tissue factor expression in endothelial cells is mediated by EGR-1.
Vascular endothelial cell growth factor (VEGF) is a major regulator of angiogenesis. We report here that treatment of endothelial cells with VEGF leads to upregulation of tissue factor mRNA and protein expression on the cell surface. Reporter gene studies show that transcriptional activation of the tissue factor gene by VEGF is mediated by a GC-rich promoter element containing overlapping binding sites for Sp1 and EGR-1. As shown by immunofluorescence and electrophoretic mobility shift assays, upon VEGF treatment EGR-1 rapidly accumulates in the nucleus and binds to its respective recognition site in the tissue factor promoter. Sp1 occupies this element in unstimulated cells and seems to be partially displaced by increasing amounts of EGR-1. Transfection of endothelial cells with an EGR-1 expression plasmid mimics the upregulation of tissue factor transcription observed after VEGF treatment. In contrast, NFkappaB, the major transcription factor involved in tissue factor upregulation by inflammatory stimuli, is not activated by VEGF. These data show that VEGF induces a response in endothelial cells largely distinct from inflammatory stimuli, and suggest that EGR-1 is a major mediator of the activation of the tissue factor and possibly other VEGF-responsive genes.
Cells, Cultured↗
Changes in the fibrinolytic system in patients with peripheral arterial occlusive disease undergoing percutaneous transluminal angioplasty.
We have investigated fibrinolytic parameters in 33 patients with peripheral arterial occlusive disease (PAOD) before and 6, 24, and 48 hours after percutaneous transluminal angioplasty (PTA) as well as in 35 gender-matched healthy controls, whose mean age was not significantly different from the mean age of the patients. PAOD patients had significantly higher plasma levels of t-PA antigen (12.0+/-4.9 vs. 9.2+/-5.5 ng/ml), PAI-1 antigen (34.8+/-22.1 vs. 27.2+/-23.6 ng/ml), PAI-1 activity (10.0+/-6.5 vs. 8.0+/-8.0 U/ml), PCI (188.2+/-55.6 vs. 134.1+/-75.5% as compared with normal human plasma), and fibrinogen (420.2+/-92.6 vs. 261.9+/-32.7 mg/dl) as compared with controls. After angioplasty, fibrinolytic parameters and fibrinogen levels increased, reaching higher than preintervention levels 24 and 48 hours after the intervention. Six months after initially successful PTA, restenosis was demonstrated in 14 out of 33 patients (42%). Patients with late restenosis had significantly higher levels of PAI-1 activity 24 and 48 hours after PTA, as compared with patients with late patency (24 hours: 16.1+/-8.0 vs. 10.0+/-7.4; 48 hours: 16.5+/-7.9 vs. 12.0+/-7.0; p<0.05 for both time points). The results suggest that impaired fibrinolysis early after PTA might be a cause or marker of a disturbed repair process of vascular injury, leading to restenosis.
Aged↗
Theileria parva 104 kDa microneme--rhoptry protein is membrane-anchored by a non-cleaved amino-terminal signal sequence for entry into the endoplasmic reticulum.
The 104 kDa microneme-rhoptry protein (p104) is the only known apical complex organelle-specific protein of Theileria parva. p104 exhibits striking structural similarities to circumsporozoite protein and sporozoite surface protein 2 of Plasmodium yoelii. Their primary sequences contain two hydrophobic segments, located at the amino-and the carboxy-terminus. The p104 amino-terminal hydrophobic region was suggested to be a signal peptide for entry into the endoplasmic reticulum and the extreme carboxy-terminal region to function as a membrane anchor. We have studied the biogenesis of p104 in a cell-free expression system and found that p104 is co-translationally transported into membranes derived from endoplasmic reticulum. The amino-terminal signal peptide is not cleaved off and anchors the protein in the membrane with the carboxy-terminal portion translocated into the lumen. We suggest that in vivo p104 is co-translationally integrated into the membrane of the endoplasmic reticulum, from where it is further transported to the microneme-rhoptry complex. Thus, p104 appears to be a suitable marker to study the development of micronemes and rhoptries in T. parva.
Animals↗
The mast cell as site of tissue-type plasminogen activator expression and fibrinolysis.
Recent data suggest that mast cells (MC) and their products (heparin, proteases) are involved in the regulation of coagulation and fibrino(geno)lysis. The key enzyme of fibrinolysis, plasmin, derives from its inactive progenitor, plasminogen, through catalytic action of plasminogen activators (PAs). In most cell systems, however, PAs are neutralized by plasminogen activator inhibitors (PAIs). We report that human tissue MC as well as the MC line HMC-1 constitutively produce, express, and release tissue-type plasminogen activator (tPA) without producing inhibitory PAIs. As assessed by Northern blotting, highly enriched lung MC (>98% pure) as well as HMC-1 expressed tPA mRNA, but did not express mRNA for PAI-1, PAI-2, or PAI-3. The tPA protein was detectable in MC-conditioned medium by Western blotting and immunoassay, and the MC agonist stem cell factor (c-Kit ligand) was found to promote the release of tPA from MC. In addition, MC-conditioned medium induced fibrin-independent plasmin generation as well as clot lysis in vitro. These observations raise the possibility that MC play an important role in endogenous fibrinolysis.
Cell Line↗
Hyperfibrinolysis in a case of myelodysplastic syndrome with leukemic spread of mast cells.
Mast cells (MC) are multipotent hemopoietic effector cells producing diverse mediators like histamine, heparin, or tissue type plasminogen activator. We report a 75-year-old male patient with myelodysplastic syndrome (MDS) of recent onset (3 months' history) associated with a massive leukemic spread of immature tryptase+ MC (tentative term: myelomastocytic leukemia). The patient presented with pancytopenia, bleeding, hypofibrinogenemia, and an increased cellular tryptase level. Moreover, an excessive elevation of plasmin-antiplasmin complexes (9,200 ng/ml; normal range: 10-150), an elevated D-dimer, and an increase in thrombin-antithrombin III complexes were found. The identity of the circulating MC was confirmed by immunophenotyping (CD117/c-kit+, CD123/IL-3R alpha-, CD11b/C3biR-), biochemical analysis (cellular ratio [ng:ng] of tryptase to histamine >1), and electron microscopy. Bone marrow (bm) examination showed trilineage dysplasia (17% blasts), 30% diffusely scattered MC, and a complex karyotype. No dense, compact MC infiltrates (mastocytosis) were detectable in bm sections. Despite hyperfibrinolysis and mediator syndrome (flushing, headache), the patient received remission induction polychemotherapy (DAV) followed by two cycles of consolidation with intermediate dose ARA-C (2 x 1 g/m2/day on days 1, 3, and 5). He entered complete remission after the first chemotherapy cycle without evidence of recurring MDS. Moreover, in response to chemotherapy, the hyperfibrinolysis and mediator syndrome resolved, and the circulating c-kit+ MC disappeared. We suggest consideration of polychemotherapy as a therapeutic option in patients with high-risk MDS of recent onset, even in the case of MC lineage involvement.
Aged↗
M6P/IGFII-receptor complexes urokinase receptor and plasminogen for activation of transforming growth factor-beta1.
Transforming growth factor-beta1 (TGF-beta1) is a critical cytokine for cell proliferation and differentiation. It is secreted by many cells in a latent pro-form (LTGF-beta1) from which biologically active TGF-beta1 is released by an in vivo mechanism that is not known. Here we show that the mannose-6-phosphate/insulin-like growth factor II-receptor (M6P/IGFII-R), which binds LTGF-beta1, complexes with urokinase (plasminogen activator)-receptor (uPA-R) on the surface of human monocytes and directly binds plasminogen (Plg). Plasmin generated from Plg in the complex mediates release of TGF-beta1 when M6P/IGFII-R is associated with uPA-R. Thus, this interaction of M6P/IGFII-R and uPA-R suggests a potential mechanism for the generation of TGF-beta1 by cells.
Amino Acid Sequence↗
Inhibition of microvascular endothelial apoptosis in tissue explants by serum albumin.
Plasma factors appear to inhibit endothelial cell (EC) apoptosis in vivo so that flow influences microvascular form. The identity of these factors has not, however, been established. Earlier, we reported that apoptosis in isolated, serum-deprived human EC is inhibited by albumin (Alb). Here, we demonstrate likely biological relevance of this to vascular remodelling in experiments with tissue explants. Rat skin explants were incubated in medium M199 with or without serum, bovine Alb, or human Alb. EC in paraffin sections of explants were labelled by lectin histochemistry and the relative proportion of apoptotic was EC determined. Apoptosis was confirmed by transmission electron microscopy and terminal deoxynucleotidyl transferase labelling. Serum-free culture induced EC apoptosis (P < 0.02) and this was strongly inhibited by Alb at physiological concentrations (P < 0.01). This was not a nonspecific protein effect, as mercaptoethanol denaturation destroyed the activity and ovalbumin was not protective. Also, protection was not due to serum contaminants, as recombinant human Alb had activity identical to that of native material. The dose response was identical for all Alb preparations tested, with maximal activity at physiological concentrations. Protection was not limited to rat tissue as similar results were obtained with human gingival explants. These data support a role for Alb as a plasma antiapoptotic factor for EC in tissues.
Animals↗
Plasmin activation system in restenosis: role in pathogenesis and clinical prediction?
During recent years it has become increasingly recognized that the plasmin activation system is involved in the development of atherosclerosis and restenosis. Responsible pathophysiologic mechanisms, however, remain elusive. This review focuses primarily on the clinicians, point of view, suggesting that increases in plasminogen activator inhibitor type-1 (PAI-1) plasma levels after balloon angioplasty or permanently elevated lipoprotein (a) (Lp(a)) plasma levels might be helpful in the prediction of restenosis after coronary angioplasty. In contrast, tissue-type plasminogen activator (tPA) plasma levels appear unrelated to restenosis, and data regarding a possible role of urokinase-type plasminogen activator (uPA) in circulation are not available at present. Furthermore, a new hypothesis on the pathophysiological role of local PAI-1 overexpression as a beneficial negative feedback mechanism to limit excess cellular proliferation in atherogenesis and restenosis is presented.
Angioplasty, Balloon, Coronary↗
Protein C inhibitor is expressed in keratinocytes of human skin.
Protein C inhibitor is a member of the serpin family that inhibits a variety of serine proteases. Protein C inhibitor is present in numerous body fluids and is produced in the liver and by various epithelial cells. To determine if this epithelial serpin is present in skin, immunohistochemical studies were performed that showed strong staining for protein C inhibitor antigen in the epidermis. Protein C inhibitor mRNA was detected in the keratinocyte cell line HaCaT and the epidermoid carcinoma cell line A431 using reverse transcription-polymerase chain reaction suggesting that also in normal skin protein C inhibitor is derived from keratinocytes. Conditioned media from these cell lines were analyzed on immunoblots, which revealed a protein C inhibitor-antigen band that comigrated with protein C inhibitor derived from the hepatoma cell line HepG2. Using an enzyme-linked immunosorbent assay specific for total protein C inhibitor antigen the accumulation of protein C inhibitor in the cell culture supernatants of HaCaT keratinocytes was found to be 0.3 ng per h per 1 million cells. This is similar to the amount of plasminogen activator inhibitor-1 produced by these cells, which also produce tissue plasminogen activator and urokinase. Fluorescence-activated cell sorter analysis revealed similar expression of intracellular protein C inhibitor antigen in proliferating and confluent HaCaT cells. These findings demonstrate that protein C inhibitor antigen is present in the normal epidermis and that protein C inhibitor is constitutively expressed by keratinocytes in culture. Therefore, protein C inhibitor may provide protease inhibitory activity not only to internal, but also to the external surface of the body. Additionally, protein C inhibitor could contribute to the regulation of retinoid supply in the epidermis, as we have shown recently that retinoic acid binds specifically to protein C inhibitor.
Cell Line↗
The role of the plasminogen activation system in cancer.
Hemostatic disorders are frequently observed in patients with malignancy with a significant proportion developing thrombotic and/or hemorrhagic complications including disseminated intravascular coagulation (DIC), deep venous thrombosis (DVT), and thrombocytopenia. Together, these abnormalities are the second most common cause of mortality in cancer patients, which has led many investigators to try to unravel the pathogenesis of thromboembolic disease, in the eventuality that this will lead to novel therapeutic treatments. The plasminogen activation system is one pathway that has been consistently implicated in cancer. Its relevance to cancer extends from being responsible for many of the hemorrhagic episodes that occur in cancer patients to being fundamental to many, if not all of the molecular mechanisms that define tumor progression. Recent developments of clinical significance shall be reviewed with respect to the role of the plasminogen activation system in tumor growth and metastasis dissemination and in the thrombophilic state in the cancer patient.
Blood Coagulation↗
Histopathological findings in experimental aneurysms embolized with conventional and thrombogenic/antithrombolytic Guglielmi coils.
We studied the short- and long-term histological responses induced by conventional and modified electronically detachable coils (GDCs) in experimental aneurysms. Eighteen carotid bifurcation aneurysms were produced microsurgically in rabbits. Six animals each were treated either with conventional or with GDCs coated with a mixture of tissue-thromboplastin to enhance intra-aneurysmal thrombus formation and of plasminogen activator inhibitor type-1 (PAI-1) in inhibit intra-aneurysmal clot fibrinolysis. Six served as untreated controls. Follow-up angiograms were obtained immediately and at 3, 6, 9, 12, 17, and 24 weeks after embolization prior to sacrifice of the animals. All aneurysms were studied macroscopically and histopathologically with the coils in situ. Five of six control aneurysms remained patent. Endovascular occlusion rates between > 90% and 100% were achieved in nine of twelve coiled aneurysms. Follow-up angiography demonstrated recanalization and coil compaction in 5 of them. Gross and microscopic histopathological examination revealed a membrane covering the orifice, intra-aneurysmal scar formation, and development of a neo-intima in both treatment groups at 17 and 24 weeks postembolization. The granulation tissue response appeared to be equally distributed in aneurysms treated with either uncoated or coated coils. Further quantitative morphometric studies are needed to prove if a thrombogenic/antithrombolytic coil-coating might be of value in providing a more enduring anatomic result after GDC-treatment of human brain aneurysms.
Aneurysm↗
Characterization of IgE-reactive autoantigens in atopic dermatitis. 1. Subcellular distribution and tissue-specific expression.
We have previously reported that patients suffering from atopic dermatitis (AD) frequently display IgE autoantibody reactivity to human proteins expressed in cell lines of different histogenetic origins. Molecular cloning and in situ staining experiments revealed that certain IgE-reactive autoantigens were expressed preferentially in target organs of atopy while others could be detected in various tissues and cell types. Here we use serum IgE from AD patients to investigate the distribution of autoantigens in subcellular fractions of the human epithelial cell line A431 and in human tissue specimens. Results obtained showed that IgE-reactive autoantigens can be detected in the nuclear > microsomal > mitochondrial > cytoplasmic fraction of A431 cells as well as in a variety of human tissue specimens (brain, bone, intestine, liver, lung, muscle, skin, uterus) and effector cells of atopy (basophils, mast cells, T cells). If IgE autoreactivity plays a pathogenetic role in severe forms of atopy, organ-specific manifestations (e.g. AD) may result from the transport to and deposition of IgE-reactive autoantigens in certain target organs (skin) rather than from a preferential expression of the autoantigens in the affected tissues.
Adult↗
Characterization of IgE-reactive autoantigens in atopic dermatitis. 2. A pilot study on IgE versus IgG subclass response and seasonal variation of IgE autoreactivity.
Previously we reported that patients with severe forms of atopy (e.g. atopic dermatitis, AD) frequently display IgE reactivity against autoantigens. Here we investigated the effects of periodate treatment and reducing versus nonreducing conditions on IgE recognition of nitrocellulose-blotted human cell extracts. IgE and IgG subclass reactivities of AD patients to blotted human cellular extracts as well as to ELISA plate-bound purified endogenous (myosin, histones) antigens and an environmental allergen (timothy grass pollen allergen, Phl p 5) were compared. Serum samples were collected over a period of 12 months from 3 autoreactive AD patients with pollen allergy and tested for IgE reactivity to nitrocellulose-blotted human epithelial and endothelial cell extracts as well as to birch and timothy grass pollen allergens. Results obtained indicate that (1) IgE autoantibodies may be directed primarily against protein and not carbohydrate epitopes, (2) reducing conditions seem to expose or better extract epitopes recognized by IgE autoantibodies, (3) IgE and IgG1-4 autoantibody responses were poorly associated and (4) IgE responses to autoallergens may reflect skin manifestations and may be boosted by seasonal exposure to pollen allergens. Our results thus indicate that IgE autoreactivity may represent a true form of autoimmunity directed against partly denatured peptide epitopes which may be boosted by exogenous allergen contact.
Adult↗