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1.
Osteoarthritis Cartilage ; 29(2): 257-268, 2021 02.
Article in English | MEDLINE | ID: mdl-33301945

ABSTRACT

OBJECTIVES: We hypothesize that chondrocytes from the deepest articular cartilage layer are pivotal in maintaining cartilage integrity and that the modification of their prehypertrophic phenotype to a hypertrophic phenotype will drive cartilage degradation in osteoarthritis. DESIGN: Murine immature articular chondrocytes (iMACs) were successively cultured into three different culture media to induce a progressive hypertrophic differentiation. Chondrocyte were phenotypically characterized by whole-genome microarray analysis. The expression of IL-34 and its receptors PTPRZ1 and CSF1R in chondrocytes and in human osteoarthritis tissues was assessed by RT-qPCR, ELISA and immunohistochemistry. The expression of bone remodeling and angiogenesis factors and the cell response to IL-1ß and IL-34 were investigated by RT-qPCR and ELISA. RESULTS: Whole-genome microarray analysis showed that iMACs, prehypertrophic and hypertrophic chondrocytes each displayed a specific phenotype. IL-1ß induced a stronger catabolic effect in prehypertrophic chondrocytes than in iMACs. Hypertrophic differentiation of prehypertrophic chondrocytes increased Bmp-2 (95%CI [0.78; 1.98]), Bmp-4 (95%CI [0.89; 1.59]), Cxcl12 (95%CI [2.19; 5.41]), CCL2 (95%CI [3.59; 11.86]), Mmp 3 (95%CI [10.29; 32.14]) and Vegf mRNA expression (95%CI [0.20; 1.74]). Microarray analysis identified IL-34, PTPRZ1 and CSFR1 as being strongly overexpressed in hypertrophic chondrocytes. IL-34 was released by human osteoarthritis cartilage; its receptors were expressed in human osteoarthritis tissues. IL-34 stimulated CCL2 and MMP13 in osteoblasts and hypertrophic chondrocytes but not in iMACs or prehypertrophic chondrocytes. CONCLUSION: Our results identify prehypertrophic chondrocytes as being potentially pivotal in the control of cartilage and subchondral bone integrity. Their differentiation into hypertrophic chondrocytes initiates a remodeling program in which IL-34 may be involved.


Subject(s)
Bone Remodeling/genetics , Chondrocytes/metabolism , Interleukins/genetics , Osteoarthritis/genetics , Aged , Aged, 80 and over , Animals , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 2/metabolism , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein 4/metabolism , Cartilage, Articular , Cell Differentiation , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Chemokine CXCL12/genetics , Chemokine CXCL12/metabolism , Chondrocytes/pathology , Female , Humans , Hypertrophy , Interleukins/metabolism , Male , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , Mice , Middle Aged , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Osteoarthritis/metabolism , Osteoarthritis/pathology , Phenotype , Receptor-Like Protein Tyrosine Phosphatases, Class 5/genetics , Receptor-Like Protein Tyrosine Phosphatases, Class 5/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism
2.
J Thromb Haemost ; 15(9): 1859-1866, 2017 09.
Article in English | MEDLINE | ID: mdl-28696550

ABSTRACT

Essentials Diagnosis of sitosterolemia, a rare recessive or syndromic disorder, is usually delayed. Peripheral blood smear is extremely useful for establishing the suspicion of sitosterolemia. High-throughput sequencing technology enables the molecular diagnosis of inherited thrombocytopenias. Accurate characterization of sitosterolemia helps us determine appropriate management. SUMMARY: Background Sitosterolemia (STSL) is a recessive inherited disorder caused by pathogenic variants in the ABCG5 and ABCG8 genes. Increased levels of plasma plant sterols (PSs) usually result in xanthomas and premature coronary atherosclerosis, although hematologic abnormalities may occasionally be present. This clinical picture is unfamiliar to many physicians, and patients may be at high risk of misdiagnosis. Objectives To report two novel ABCG5 variants causing STSL in a Spanish patient, and review the clinical and mutational landscape of STSL. Patient/Methods A 46-year-old female was referred to us with lifelong macrothrombocytopenia. She showed familial hypercholesterolemia-related xanthomas. Molecular analysis was performed with high-throughput sequencing. Plasma PS levels were evaluated with gas-liquid chromatography. The STSL landscape was reviewed with respect to specific online databases and all reports published since 1974. Results A blood smear revealed giant platelets and stomatocytes. Novel compound heterozygous variants were detected in exons 7 (c.914C>G) and 13 (c.1890delT) of ABCG5. The patient showed an increased plasma level of sitosterol. These findings support the diagnosis of STSL. In our review, we identified only 25 unrelated STLS patients who presented with hematologic abnormalities including macrothrombocytopenia. It remains unknown why only some patients develop hematologic abnormalities. Conclusions This is the first Spanish STSL patient to be reported and molecularly characterized. The early diagnosis of STLS is strongly supported by the presence of stomatocytes in blood smears. The definitive diagnosis of STSL by measurement of serum PS levels and molecular analyses prompted the use of ezetimibe therapy.


Subject(s)
ATP Binding Cassette Transporter, Subfamily G, Member 5/genetics , Hypercholesterolemia/genetics , Intestinal Diseases/genetics , Lipid Metabolism, Inborn Errors/genetics , Lipoproteins/genetics , Mutation , Phytosterols/adverse effects , Thrombocytopenia/genetics , Xanthomatosis/genetics , Anticholesteremic Agents/therapeutic use , DNA Mutational Analysis , Ezetimibe/therapeutic use , Female , Genetic Predisposition to Disease , High-Throughput Nucleotide Sequencing , Humans , Hypercholesterolemia/blood , Hypercholesterolemia/diagnosis , Hypercholesterolemia/drug therapy , Intestinal Diseases/blood , Intestinal Diseases/diagnosis , Intestinal Diseases/drug therapy , Lipid Metabolism, Inborn Errors/blood , Lipid Metabolism, Inborn Errors/diagnosis , Lipid Metabolism, Inborn Errors/drug therapy , Middle Aged , Phenotype , Phytosterols/blood , Phytosterols/genetics , Sitosterols/blood , Spain , Thrombocytopenia/blood , Thrombocytopenia/diagnosis , Xanthomatosis/blood , Xanthomatosis/diagnosis
3.
Haemophilia ; 22(4): 590-7, 2016 Jul.
Article in English | MEDLINE | ID: mdl-26879396

ABSTRACT

INTRODUCTION: Molecular testing of Inherited bleeding coagulation disorders (IBCDs) not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and in certain cases genotype-phenotype correlations are important for predicting the clinical course of the disease and to allow tailor-made follow-up of individuals. Until recently, genotyping has been mainly performed by Sanger sequencing, a technique known to be time consuming and expensive. Currently, next-generation sequencing (NGS) offers a new potential approach that enables the simultaneous investigation of multiple genes at manageable cost. AIM: The aim of this study was to design and to analyse the applicability of a 23-gene NGS panel in the molecular diagnosis of patients with IBCDs. METHODS: A custom target enrichment library was designed to capture 31 genes known to be associated with IBCDs. Probes were generated for 296 targets to cover 86.3 kb regions (all exons and flanking regions) of these genes. Twenty patients with an IBCDs phenotype were studied using NGS technology. RESULTS: In all patients, our NGS approach detected causative mutations. Twenty-one pathogenic variants were found; while most of them were missense (18), three deletions were also identified. Six novel mutations affecting F8, FGA, F11, F10 and VWF genes, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant. CONCLUSION: Our results demonstrate that this approach could be an accurate, reproducible and reliable tool in the rapid genetic diagnosis of IBCDs.


Subject(s)
Blood Coagulation Disorders, Inherited/genetics , Genetic Testing/methods , Adolescent , Adult , Blood Coagulation Disorders, Inherited/pathology , Child , Child, Preschool , DNA/chemistry , DNA/isolation & purification , DNA/metabolism , Female , Frameshift Mutation , Gene Deletion , Genetic Association Studies , Genotype , High-Throughput Nucleotide Sequencing , Humans , Infant , Male , Middle Aged , Mutation, Missense , Sequence Analysis, DNA , Young Adult
4.
Thromb Haemost ; 114(2): 313-24, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25994029

ABSTRACT

Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbß3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbß3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmann's thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.


Subject(s)
Hemorrhagic Disorders/immunology , Immunoglobulin M/immunology , Platelet Aggregation/immunology , Platelet Membrane Glycoproteins/physiology , Thrombocytopenia/immunology , Adult , Afibrinogenemia/blood , Autoantibodies/blood , Biomarkers/blood , Cold Temperature/adverse effects , Cryoglobulins/pharmacology , Female , Humans , Platelet Activation/immunology , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Protein-Tyrosine Kinases/blood , Thrombasthenia/blood , Thrombocytopenia/blood
5.
Hamostaseologie ; 34(4): 301-9, 2014.
Article in English | MEDLINE | ID: mdl-25117010

ABSTRACT

Hermansky-Pudlak syndrome (HPS) is a rare, autosomal recessive disorder affecting lysosome-related organelles (LRO), including dense platelet granules. HPS causes oculocutaneous hypopigmentation, bleeding diathesis and granulomatous colitis or pulmonary fibrosis. To date, there is no curative treatment and the clinical management depends on the severity of symptoms. A prompt diagnosis of HPS patients could improve their quality of life and clinical management. However, the absence of a specific platelet function test, the wide molecular heterogeneity, and the lack of phenotype-genotype correlations hamper the rapid diagnosis. Nine subtypes of HPS have been identified as a result of mutations in nine genes that codify for proteins involved in formation and shuttle of the LRO. The molecular characterization of patients and knowledge derived from animal models of HPS contribute to the understanding of biogenesis and function of the LRO. This paper describes a patient with a novel homozygous nonsense mutation causing HPS and provides a review of the literature focusing on recent advances in the molecular characterization and physiopathology of HPS.


Subject(s)
Genetic Predisposition to Disease/genetics , Hermanski-Pudlak Syndrome/diagnosis , Hermanski-Pudlak Syndrome/genetics , Membrane Proteins/genetics , Molecular Diagnostic Techniques/methods , Polymorphism, Single Nucleotide/genetics , Adult , Base Sequence , Evidence-Based Medicine , Genetic Markers/genetics , Genetic Testing/methods , Genetic Variation/genetics , Humans , Male , Molecular Sequence Data
7.
Vox Sang ; 107(4): 343-50, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24916720

ABSTRACT

BACKGROUND AND OBJECTIVES: Inaccuracy of fingerstick haemoglobin compromises donor's health and losses blood donations. We evaluated the benefit of double haemoglobin screening with HemoCue. STUDY DESIGN AND METHODS: Blood donors underwent fingerstick screening by HemoCue and were driven for donation if capillary haemoglobin was within the regulatory range. Those failing were drawn venous blood and donated if their venous haemoglobin determined with HemoCue was acceptable. RESULTS: Of 276 605 donor clinic visits, 10 011 (3·6%) were assessed by two-step haemoglobin screening using HemoCue, because of low (n = 9444) or high (n = 567) capillary haemoglobin. Among these, 2561 (25·6%) were deemed eligible [recovered donations]. The recovery rate was 23·8% and 55·0% among donors presenting with low and high capillary haemoglobin, respectively. In both categories of attempted donations, capillary and venous haemoglobin with HemoCue correlated significantly in recovered donors (R(2)  ≈ 0·5-0·7) but not in deferred visits (R(2)  < 0·15). Venous haemoglobin with HemoCue and by haematological analyzer significantly correlated in all donations attempts (R(2)  ≈ 0·7). Donors presenting with low capillary haemoglobin showed small bias between capillary and venous haemoglobin by HemoCue (-2·4 ± 6·2 g/l), fingerstick haemoglobin and venous haemoglobin with counter (1·3 ± 7·3 g/l), and venous haemoglobin with HemoCue and counter (3·7 ± 3·9 g/l). This bias was slightly greater in donors with high capillary haemoglobin (-7·5 ± 7·8, 13·7 ± 7·5, and 6·2 ± 7·5, respectively). Double haemoglobin screening by HemoCue reached an accuracy of 87·3% for qualifying donors presenting with low fingerstick haemoglobin. CONCLUSIONS: Double haemoglobin measurement with HemoCue [fingerstick and venous blood if required] is feasible and allows a significant recovery of blood donations.


Subject(s)
Blood Specimen Collection/methods , Hemoglobinometry , Hemoglobins/analysis , Adult , Aged , Blood Donors , Blood Specimen Collection/instrumentation , Donor Selection , Female , Hemoglobinometry/instrumentation , Humans , Male , Odds Ratio
8.
Stem Cells ; 32(2): 436-46, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24420904

ABSTRACT

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2(Δ) (27/) (Δ27)), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2(Δ) (27/) (Δ27) induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2(Δ) (27/) (Δ27) iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2(Δ) (27/) (Δ27) mouse embryonic fibroblasts. Gene-corrected Brca2(Δ) (27/) (Δ27) iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2(Δ) (27/) (Δ27) recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2(Δ) (27/) (Δ) (27) iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed.


Subject(s)
BRCA2 Protein/genetics , Fanconi Anemia/genetics , Genetic Therapy , Hematopoietic Stem Cells , Induced Pluripotent Stem Cells/cytology , Animals , BRCA2 Protein/biosynthesis , Cell Differentiation/genetics , Cells, Cultured , Cellular Reprogramming , DNA Damage/genetics , Fanconi Anemia/pathology , Fanconi Anemia/therapy , Fibroblasts/metabolism , Hematopoietic Stem Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , Mice
9.
Thromb Res ; 133(3): 412-7, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24388574

ABSTRACT

UNLABELLED: PMM2-CDG, the most frequent congenital disorder of N-glycosylation, is an autosomal recessive disease with a multisystem presentation. PMM2-CDG patients show an increased risk for thrombosis, which might be in part due to spontaneous platelet aggregations as previously described. A potential hypoglycosylation of platelet proteins in these patients might explain this increased reactivity, as removal of sialic acid from platelets, particularly of GPIbα, leads to enhance platelet aggregation and clearance from the circulation. This study is the first one that has evaluated the glycosylation status of platelet proteins in 6 PMM2-CDG patients using different approaches including immunoblot, RCA120 lectin binding to platelets and expression of different membrane platelet N-glycoproteins by flow cytometry, as well as by platelet N-glycoproteome analysis. RCA120 lectin binding to the platelet membrane of PMM2-CDG patients showed evidence for decreased sialic acid content. However, immunoblot and flow cytometric analysis of different platelet N-glycoproteins, together with the more sensitive 2D-DIGE analysis, suggest that platelet N-glycoproteins, including GPIbα, seem to be neither quantitatively nor qualitatively significantly affected. The increased binding of RCA120 lectin could be explained by the abnormal glycosylation of hepatic proteins being attached to the platelets. CONCLUSIONS: This is the first study that has evaluated the platelet N-glycoproteome. Our findings suggest that platelet proteins are not significantly affected in PMM2-CDG patients. Further studies are still warranted to unravel the mechanism(s) that increase(s) the risk of thrombosis in these patients.


Subject(s)
Congenital Disorders of Glycosylation/blood , Platelet Membrane Glycoproteins/analysis , Case-Control Studies , Glycosylation , Humans , Platelet Membrane Glycoproteins/metabolism , Proteomics
10.
Vox Sang ; 106(1): 38-44, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23888911

ABSTRACT

BACKGROUND AND OBJECTIVES: Buffy-coat (BC)-derived platelet concentrates (PCs) are the predominant product for platelet transfusion in many countries. Two automated systems, OrbiSac and TACSI, have been introduced in blood centres to prepare these PCs, as an alternative to the manual method. We compared the in vitro quality of PCs prepared by both methods during standard storage. STUDY DESIGN AND METHODS: Twenty primary BC pools were split into two parts, which were processed with OrbiSac and TACSI system to obtain OrbiSac PCs (O-PCs) and TACSI PCs (T-PCs), respectively. On days 1, 5 and 7 of standard storage, samples were taken and the following analysed: cell count, metabolic variables, platelet function and content of activation and proinflammatory substances. RESULTS: Both the OrbiSac and TACSI systems produced PCs that meet the standards for platelet products in terms of platelet and leucocyte content. In vitro evaluation pointed to the similar preservation of platelet metabolism (pH, glucose, bicarbonate and lactate) in O-PCs and T-PCs. Moreover, there were no significant differences between O-PCs and T-PCs as regards the hypotonic shock response or in the platelet aggregation profile. The OrbiSac system caused greater platelet activation, which resulted in higher concentrations of sCD62P, RANTES and sCD40L on the day the PCs were prepared. CONCLUSION: The systems OrbiSac and TACSI can be used to produce buffy-coat-derived PCs whose cell content, platelet function and metabolism are similar during standard storage. However, the preparation with the OrbiSac system induces a transient increase in platelet activation and release of proinflammatory substances.


Subject(s)
Blood Buffy Coat/cytology , Blood Platelets/cytology , Plasma/cytology , Plateletpheresis/instrumentation , Blood Buffy Coat/physiology , Blood Platelets/physiology , Humans , Leukocyte Reduction Procedures , Plasma/chemistry , Platelet Activation , Platelet Aggregation , Platelet Function Tests , Platelet Transfusion
11.
Thromb Haemost ; 105(5): 855-63, 2011 May.
Article in English | MEDLINE | ID: mdl-21384078

ABSTRACT

ß1-tubulin is the main constituent of the platelet marginal band and studies with deficient mice showed that it maintains discoid shape and it is required for normal platelet formation. TUBB1 Q43P polymorphism is associated with decreased ß1-tubulin expression, diminished platelet reactivity, and partial loss of discoid shape in heterozygous carriers. However, to date no studies have been carried out on homozygous PP individuals. Our study included 19 subjects genotyped for TUBB1 Q43P polymorphism (4 QQ, 4 QP, and 2 PP). The two PP individuals were recruited after genotyping of 2073 individuals. Biochemical, microscopy, and molecular studies were performed. Real-time PCR showed a ~40% decrease in TUBB1 mRNA in the two PP individuals compared to four QQ subjects. Western blot analysis confirmed this reduction. Electron microscopy revealed a majority of normal discoid platelets in PP individuals, although platelets with loose, re-orientated or invaginated protofilaments, and an over-developed open canalicular system were observed. Such abnormalities were not observed in QQ subjects. Morphometric analyses showed no differences between PP and QQ individuals. Immunofluorescence confirmed the presence of a normal marginal band in a majority of platelets from PP subjects. Interestingly, both PP subjects had a 40% lower platelet count than QP and QQ. TUBB1 Q43P polymorphism in homozygosity mildly affects platelet ultrastructure and our data further suggest that high levels of ß1-tubulin might not be critical to sustain platelet discoid shape.


Subject(s)
Blood Platelets/ultrastructure , Platelet Activation , Tubulin/metabolism , Adult , Blood Platelets/metabolism , Cell Count , Cytoskeleton/ultrastructure , DNA Mutational Analysis , Female , Homozygote , Humans , Male , Microscopy, Electron , Middle Aged , Platelet Activation/genetics , Polymorphism, Genetic , Tubulin/genetics
12.
Br J Pharmacol ; 158(6): 1548-56, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19814731

ABSTRACT

BACKGROUND AND PURPOSE: The modulation by flavonoids of platelet responses induced by thrombin has been little investigated, and the antiplatelet activity, as well as possible inhibitory mechanisms of these compounds on thrombin signalling, has not yet been elucidated. We explored whether flavonoids affect platelet signalling pathways triggered by thrombin and by the selective activation of its protease-activated receptors (PARs) 1 and 4, and analysed the antagonism of these polyphenols at thrombin receptors. EXPERIMENTAL APPROACH: We investigated the effect of a range of polyphenolic compounds on platelet aggregation, 5-HT secretion, intracellular calcium mobilization, protein kinase activity and tyrosine phosphorylation, triggered by thrombin and PAR agonist peptides (PAR-APs). The ability of these flavonoids to bind to thrombin receptors was investigated by competitive radioligand binding assays using (125)I-thrombin. KEY RESULTS: Quercetin, apigenin and genistein impaired platelet aggregation, as well as 5-HT release and calcium mobilization, induced by thrombin and PAR-APs. Quercetin and apigenin were inhibitors of protein kinases, but genistein exhibited a minimal ability to suppress platelet phosphorylation. Binding assays did not establish any kind of interaction between thrombin receptors and any of the flavonoids tested. CONCLUSIONS AND IMPLICATIONS: Quercetin, apigenin and genistein did not inhibit thrombin responses by interacting with thrombin receptors, but by interfering with intracellular signalling. While inhibition by genistein may be a consequence of affecting calcium mobilization, subsequent platelet secretion and aggregation, for quercetin and apigenin, inhibition of kinase activation may also be involved in the impairment of platelet responses.


Subject(s)
Apigenin/pharmacology , Genistein/pharmacology , Platelet Aggregation/drug effects , Quercetin/pharmacology , Calcium/metabolism , Flavonoids/pharmacology , Humans , Platelet Aggregation Inhibitors/pharmacology , Radioligand Assay , Receptor, PAR-1/drug effects , Receptor, PAR-1/metabolism , Receptors, Thrombin/drug effects , Receptors, Thrombin/metabolism , Serotonin/metabolism , Signal Transduction/drug effects , Thrombin/metabolism
13.
J Inherit Metab Dis ; 32 Suppl 1: S265-7, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19562501

ABSTRACT

The frequency of monoclonal gammopathy of undetermined significance (MGUS) is higher in patients with type I Gaucher disease (GD I) than in the general population. Although enzyme replacement therapy is effective in the control of the disease, its effect on MGUS is still controversial. We present the case of a 65-year-old woman with extensive GD I associated with IgM MGUS, in whom enzyme replacement therapy succeeded in eradicating the monoclonal component. This observation further supports the idea that enzyme replacement therapy decreases the chronic antigenic stimulation responsible for gammopathies in Gaucher disease.


Subject(s)
Enzyme Replacement Therapy , Gaucher Disease/complications , Gaucher Disease/drug therapy , Glucosylceramidase/therapeutic use , Immunoglobulin M/blood , Monoclonal Gammopathy of Undetermined Significance/etiology , Monoclonal Gammopathy of Undetermined Significance/immunology , Aged , Antibodies, Monoclonal/blood , Female , Gaucher Disease/immunology , Glucosylceramidase/blood , Glucosylceramidase/genetics , Humans , Recombinant Proteins/therapeutic use
14.
J Agric Food Chem ; 56(9): 2970-6, 2008 May 14.
Article in English | MEDLINE | ID: mdl-18410117

ABSTRACT

Previous studies using washed platelets demonstrated that certain flavonoids inhibit platelet function through several mechanisms including blockade of TxA(2) receptors (TPs). We aimed to analyze the binding capacity of flavonoids to TPs in platelet rich plasma (PRP), investigated their effect in flowing blood, and evaluated the ability of apigenin to improve the efficacy of aspirin in the inhibition of platelet aggregation. The binding of flavonoids to TPs in PRP was explored using binding assays and the TP antagonist [ (3)H]SQ29548. Effects of flavonoids on platelet adhesion were assessed using arterial subendothelium with annular plate perfusion chambers, and global evaluation of apigenin on high-shear-dependent platelet function was determined by the PFA-100. To evaluate the ability of apigenin to potentiate the effect of aspirin, arachidonic acid-induced platelet aggregation was measured prior to and after consumption of subaggregatory doses of aspirin in the presence or absence of apigenin. Binding assays revealed that apigenin was an efficient competitor of [ (3)H]SQ29548 binding to PRP ( K i = 155.3 +/- 65.4 microM), and perfusion studies showed that apigenin, genistein, and catechin significantly diminished thrombus formation when compared to control (26.2 +/- 3.8, 33.1 +/- 5.2, and 26.2 +/- 5.2 vs 76.6 +/- 2.6%, respectively; p < 0.05). Apigenin, similarly to the TP antagonist SQ29548, significantly prolonged collagen epinephrine-induced PFA-100 closure time in comparison to the control and, when added to platelets that had been exposed in vivo to aspirin, potentiated its inhibitory effect on platelet aggregation. The inhibitory effect of some flavonoids in the presence of plasma, particularly apigenin, might in part rely on TxA(2) receptor antagonism. There is a clear increase in the ex vivo antiplatelet effect of aspirin in the presence of apigenin, which encourages the idea of the combined use of aspirin and certain flavonoids in patients in which aspirin fails to properly suppress the TxA(2) pathway.


Subject(s)
Apigenin/pharmacology , Arachidonic Acid/metabolism , Aspirin/pharmacology , Platelet Adhesiveness/drug effects , Platelet Aggregation Inhibitors/pharmacology , Apigenin/metabolism , Blood Platelets/drug effects , Blood Platelets/physiology , Bridged Bicyclo Compounds, Heterocyclic , Drug Synergism , Endothelium/physiology , Fatty Acids, Unsaturated , Humans , Hydrazines/metabolism , Platelet Aggregation/drug effects , Platelet-Rich Plasma/metabolism , Receptors, Thromboxane A2, Prostaglandin H2/antagonists & inhibitors , Receptors, Thromboxane A2, Prostaglandin H2/metabolism , Thrombosis
16.
Int J Artif Organs ; 28(2): 164-9, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15770593

ABSTRACT

The preservation of the peritoneal membrane is crucial for long-term survival in peritoneal dialysis. Epithelial-to-mesenchymal transition (EMT) is a process demonstrated in mesothelial cells (MC), responsible for negative peritoneal changes and directly related to PD. EMT enables neovascularization and fibrogenic capabilities in MC. Vascular endothelial growth factor (VEGF) is the mediator for neo-vascularization. Rapamycin is a potent immunosuppressor with antifibrotic action in renal allografts and has a demonstrated anti-VEGF effect. We performed this study with the hypothesis that rapamycin may regulate the EMT of MC. MC from human omentum were cultured. When mesothelial cells reached confluence, some of them were stimulated with r-TGF-beta (1 ng/mL) to induce EMT, co-administered with rapamycin (0.2, 2, 4, 20 and 40 nM). Other groups of cells received similar doses of rapamycin or r-TGF-beta, separately. Cells were analyzed at 6, 24, 48 hours and 7 days. As markers of EMT we included alfa -SMA, E-cadherin and snail nuclear factor by quantitative RT-PCR. EMT markers and regulators demonstrated the following changes with rapamycin: E-cadherin (a protective gene for EMT) increased 2.5-fold relative to controls under 40 nM, at 24h. Importantly, rapamycin inhibited snail expression induced by TGF-beta at 6h, whereas TGF-beta increased snail 10-fold. At day 7, rapamycin showed no anti-EMT properties. An important decrease in alfa -SMA expression by MC after rapamycin addition was observed. In conclusion, rapamycin shows a mild protective effect on EMT, as it increases E-cadherin and decreases alfa -SMA expression. Consequently, rapamycin might partially regulate the epithelial-to-mesenchymal transition of mesothelial cells.


Subject(s)
Epithelial Cells/drug effects , Epithelial Cells/metabolism , Immunosuppressive Agents/pharmacology , Omentum/cytology , Sirolimus/pharmacology , Actins/metabolism , Biomarkers/metabolism , Blotting, Western , Cadherins/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression/drug effects , Humans , In Vitro Techniques , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta/pharmacology
17.
Int J Artif Organs ; 28(2): 105-11, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15770598

ABSTRACT

OBJECTIVE: To evaluate the utility of peritoneal pathologic samples, unrelated to peritoneal dialysis (PD) treatment, for the study of peritoneal fibrosis and inflammation. METHODS: Comparative morphologic and immunohistochemical study of peritoneal pathologic samples unrelated to PD with peritoneal biopsies from PD patients with special emphasis on the expression of myofibroblastic and epithelial-to-mesenchymal transition markers. RESULTS: Regarding morphology, PD-related simple fibrosis was less cellular, with greater stromal hyalinization, determining a homogeneous, hypocellular aspect of the submesothelium. In contrast, non-PD fibrosis was more cellular with an extracellular matrix showing a dense and fibrillar quality with wide bundles of collagen. Hylinazing vasculopathy was only present in PD samples. Myofibroblastic differentiation and epithelial-to-mesenchymal transition were common findings in all situations of peritoneal fibrosis. Calponin and calretinin are useful cellular markers to study such fibrogenic mechanisms and correlate with other well-known markers such as a -SMA and cytokeratins. Their expression was much more intense in those samples showing acute inflammation (peritonitis). CONCLUSIONS: Non-PD models of peritoneal fibrosis seem very useful to evaluate important features of human peritoneal pathology such us fibrogenesis, and inflammation. Fibrogenic events such as myofibroblastic differentiation and epithelial-to-mesenchymal transition are evident in these tissue samples allowing us to use them as an accessible source for in vivo and ex vivo studies. Both events show their maximal expression in situations of acute inflammation supporting the important role that peritonitis episodes play in the progression of fibrosis.


Subject(s)
Epithelium/metabolism , Epithelium/pathology , Peritoneum/pathology , Actins/metabolism , Biomarkers , Biopsy , Calbindin 2 , Calcium-Binding Proteins/metabolism , Case-Control Studies , Cell Differentiation , Edema/pathology , Fibrin/metabolism , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Hernia, Inguinal/metabolism , Hernia, Inguinal/pathology , Humans , Hyalin/metabolism , Keratins/metabolism , Microfilament Proteins , Neutrophils/metabolism , S100 Calcium Binding Protein G/metabolism , Sclerosis , Tissue Adhesions/metabolism , Tissue Adhesions/pathology , Calponins
18.
J Thromb Haemost ; 3(2): 369-76, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15670046

ABSTRACT

BACKGROUND: Dietary flavonoids are known for their antiplatelet activity resulting in cardiovascular protection, although the specific mechanisms by which this inhibition occurs has not been fully established. OBJECTIVE: The aim of this study was to investigate the interaction of nine flavonoids representative of various chemical classes, with platelet responses dependent on thromboxane A(2) (TxA(2)) generation and on receptor antagonism, and to analyze the structural requirements for such effects. METHODS: The effect of several types of flavonoids on platelet aggregation, serotonin release, and TxA(2) generation was investigated. Competitive radioligand binding assays were used to screen for affinity of these compounds to TxA(2) receptors. RESULTS: Flavones (apigenin and luteolin) and isoflavones (genistein) abrogated arachidonic acid and collagen-induced platelet responses, such as aggregation and secretion, with a less substantial effect on TxA(2) synthesis. These compounds were identified as specific ligands of the TxA(2) receptor in the micromol L(-1) range, this effect accounting for antiplatelet effects related to stimulation with those agonists. Tight binding of flavonoids to the human TxA(2) receptor relies on structural features such as the presence of the double bond in C2-C3, and a keto group in C4. CONCLUSIONS: The inhibition by specific flavonoids of in vitro platelet responses induced by collagen or arachidonic acid seems to be related, to a great extent, to their ability to compete for binding to the TxA(2) receptor. Therefore, antagonism of this TxA(2) receptor may represent an additional mechanism for the inhibitory effect of these compounds in platelet function.


Subject(s)
Flavonoids/pharmacology , Platelet Aggregation/drug effects , Receptors, Thromboxane A2, Prostaglandin H2/metabolism , Apigenin/pharmacology , Genistein/pharmacology , Humans , Ligands , Luteolin/pharmacology , Platelet Aggregation Inhibitors/chemistry , Protein Binding , Receptors, Thromboxane A2, Prostaglandin H2/antagonists & inhibitors , Serotonin/metabolism , Structure-Activity Relationship , Thromboxane A2/biosynthesis
19.
Clin Lab Haematol ; 26(4): 275-86, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15279665

ABSTRACT

With the goal of producing haemostatically effective platelet concentrates (PCs) with a longer shelf-life, we aimed to identify a simple combination of platelet inhibitors, with a low pharmacological load, which could avoid the unacceptable loss of platelets stored under refrigerated conditions. PCs stored with different combinations of second messenger effectors were analysed at days 5, 10 and 15 of storage and compared with those supplemented with ThromboSol--a combination of six platelet inhibitors that protects cells from cold damage. The following parameters were analysed: platelet counts, biochemical parameters (glucose, pH, bicarbonate, lactate), cell lysis (lactic dehydrogenase, LDH), membrane glycoproteins (GPs), platelet aggregation, fibrinogen binding and hypotonic shock response. We characterized the combination of amiloride and sodium nitroprusside (at 1/2 the dose included in ThromboSol). This was found to be similar to ThromboSol and superior to nontreated units in the prevention of cold-induced platelet aggregation at day 15 of storage (maintenance of 78% and 80% of initial platelet counts, respectively), preservation of GPIbalpha (11% and 12% better maintenance of mean fluorescence intensity compared with control units, respectively), and reduced cell lysis (13% and 11% decrease in supernatant LDH, respectively). The reduced pharmacological load with the identified solution compared with ThromboSol is an argument in favour of the potential use of these agents when designing strategies to improve PC storage.


Subject(s)
Blood Platelets/drug effects , Blood Preservation/methods , Cryoprotective Agents/pharmacology , Second Messenger Systems , Blood Platelets/physiology , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Platelet Aggregation/drug effects , Platelet Count , Refrigeration , Temperature
20.
Transfus Med ; 13(5): 259-66, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14617336

ABSTRACT

Recent studies have shown that total hepatitis C virus (HCV) core antigen, both free and antibody bound, is an accurate indirect marker of viral replication that can be used in clinical practice. The aim of the present study was to evaluate the performance of a new total HCV core antigen enzyme-linked immunosorbent assay (ELISA) for detection and quantification of total core antigen in blood donors, testing positive for anti-HCV antibodies and for prospective low-risk population screening. A population comprising 257 samples, from blood donors detected reactive for anti-HCV antibodies [137 recombinant immunoblot assay (RIBA) positive and 120 RIBA indeterminate], were tested by using a new total HCV core antigen ELISA. HCV-RNA was quantified by using quantitative polymerase chain reaction (PCR) assays in all RIBA-positive samples and RIBA-indeterminate samples that were positive for the total core antigen. Specificity of the assay was studied in 1070 healthy blood donors negative for anti-HCV antibodies. Compared with quantitative PCR assays, the total HCV core antigen assay showed 97.37% sensitivity. The three HCV-RNA-positive samples, which tested negative for the total core antigen, had a low viral load (< 1.4 x 10(4) IU mL(-1)). All samples with more than 1.4 x 10(4) IU mL(-1) of viral RNA were positive for total core antigen, independent of the HCV genotype. Concentration of total core antigen correlated significantly with those of HCV-RNA (r = 0.614, P < 0.0001). Overall specificity in freshly collected blood donor specimens was 99.63%. Our data indicate that the total HCV core antigen ELISA has a sensitivity close to PCR assays in diagnosing HCV infection in blood donors with anti-HCV antibodies and shows an excellent specificity in volunteer donors. This assay, in combination with anti-HCV antibodies screening tests, could be an alternative to molecular assays for HCV infection screening in blood donors.


Subject(s)
Blood Donors , Enzyme-Linked Immunosorbent Assay/methods , Hepatitis C Antigens/blood , Enzyme-Linked Immunosorbent Assay/standards , Hepatitis C/diagnosis , Humans , Mass Screening/methods , Polymerase Chain Reaction/standards , RNA, Viral/blood , Sensitivity and Specificity , Viral Core Proteins/blood , Viral Load/methods
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