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ABSTRACT: CD38 (also known as T10) was identified in the late 1970s in the course of pioneering work carried out at the Dana-Farber Cancer Center (Boston, MA) that focused on the identification of surface molecules involved in antigen recognition. CD38 was initially found on thymocytes and T lymphocytes, but today we know that the molecule is found throughout the immune system, although its expression levels vary. Because of this, CD38 was considered an "activation marker," a term still popular in routine flow cytometry. This review summarizes the findings obtained from different approaches, which led to CD38 being re-defined as a multifunctional molecule. CD38 and its homologue CD157 (BST-1), contiguous gene duplicates on human chromosome 4 (4p15), are part of a gene family encoding products that modulate the social life of cells by means of bidirectional signals. Both CD38 and CD157 play dual roles as receptors and ectoenzymes, endowed with complex activities related to signaling and cell homeostasis. The structure-function analysis presented here is intended to give clinical scientists and flow cytometrists a background knowledge of these molecules. The link between CD38/CD157 and human diseases will be explored here in the context of chronic lymphocytic leukemia, myeloma and ovarian carcinoma, although other disease associations are also known. Thus CD38 and CD157 have evolved from simple leukocyte activation markers to multifunctional molecules involved in health and disease. Future tasks will be to explore their potential as targets for in vivo therapeutic interventions and as regulators of the immune response. © 2013 International Clinical Cytometry Society.
Cytometry Part B Clinical Cytometry 04/2013; · 2.53 Impact Factor
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Simona Morone,
Nicola Lo-Buono,
Rossella Parrotta,
Alice Giacomino,
Giulia Nacci,
Alfredo Brusco,
Alexey Larionov,
Paola Ostano,
Maurizia Mello-Grand,
Giovanna Chiorino,
Erika Ortolan, Ada Funaro
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ABSTRACT: Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little or no prospect of cure. Despite advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, unraveling the biological mechanisms underpinning EOC progression is critical for improving patients' survival. Recently, we reported that CD157 (an ectoenzyme regulating leukocyte diapedesis) is expressed in EOC and that high expression of the molecule is negatively correlated with the disease outcome in patients. Here, we demonstrate that forced overexpression of CD157 in OVCAR-3, TOV-21G, A2780 and OV-90 ovarian cancer cell lines promotes morphological and phenotypic changes characterized by disruption of intercellular junctions, downregulation of epithelial markers and upregulation of mesenchymal ones. These changes in cell shape and phenotype bring to reduced sensitivity to anoikis, increased anchorage-independent growth, cell motility and mesothelial invasion. Conversely, knockdown of CD157 in OV-90 and OC314 cells reverts the mesenchymal phenotype and reduces the cells' migratory potential. Transcriptome profiling analysis highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing OVCAR-3 and OV-90 cells. The modulation of selected genes translates into alteration of protein expression that give cells a highly malignant phenotype. The overall picture deduced from the analysis of the modulated transcripts is that high expression of CD157 strengthens a number of biological processes favoring tumor progression (including development and cell motility), and weakens several biological processes hindering tumor progression (such as apoptosis, cell death and response to stress). Together, these findings implicate CD157 in the progression of EOC to metastatic disease and suggest that CD157 may represent a valuable therapeutic target. Copyright: ß 2012 Morone et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PLoS ONE 08/2012; · 4.09 Impact Factor
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02/2012; , ISBN: 978-953-307-812-0
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ABSTRACT: CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β(1) and β(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.
Journal of Biological Chemistry 05/2011; 286(21):18681-91. · 4.77 Impact Factor
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ABSTRACT: Germline and somatic activating mutations in the LH receptor (LHR) gene have been reported.
Our objective was to perform mutation analysis of the LHR gene of patients with Leydig cell adenoma or hyperplasia. Functional studies were conducted to compare the D578H-LHR mutant with the wild-type (WT)-LHR and the D578G-LHR mutant, a classic cause of testotoxicosis. The three main signal transduction pathways in which LHR is involved were studied.
We describe eight male patients with gonadotropin-independent precocious puberty due to Leydig cell adenoma or hyperplasia.
The D578H-LHR mutation was found in the adenoma or nodule with hyperplasia in all but two patients. D578H-LHR displayed a constitutively increased but noninducible production of cAMP, led to a very high production of inositol phosphates, and induced a slight phosphorylation of p44/42 MAPK in the absence of human chorionic gonadotropin. The D578G-LHR showed a response intermediate between WT-LHR and the D578H-LHR. Subcellular localization studies showed that the WT-LHR was almost exclusively located at the cell membrane, whereas the D578H-LHR showed signs of internalization. D578H-LHR was the only receptor to colocalize with early endosomes in the absence of human chorionic gonadotropin.
Although several LHR mutations have been reported in testotoxicosis, the D578H-LHR mutation, which has been found only as a somatic mutation, appears up until now to be specifically responsible for Leydig cell adenomas. This is reflected by the different activation of the signal transduction pathways, when compared with the WT-LHR or D578G-LHR, which may explain the tumorigenesis in the D578H mutant.
The Journal of clinical endocrinology and metabolism 04/2011; 96(7):E1197-205. · 6.50 Impact Factor
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[show abstract]
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ABSTRACT: CD157, a member of the CD38 gene family, is a NAD-metabolizing ectoenzyme and a signalling molecule whose role in polarization,
migration and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain
to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and
adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration
and adhesion to fibronectin and fibrinogen, but that CD157 cross-linking is sufficient to overcome the block, suggesting an
active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the
detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β1 and β2 integrin, which,
in turn, leads to the formation of a multimolecular complex favouring signal transduction. This functional cross-talk with
integrins allows CD157 to act as a receptor, despite its intrinsic structural inability to do so on its own. Intracellular
signals mediated by CD157 rely on the integrin/Src/FAK pathway, resulting in increased activity of the MAPK-ERK1/2 and the
PI3K/Akt downstream signalling pathways, which are crucial in the control of monocyte transendothelial migration.
Collectively, these findings indicate that CD157 acts as a molecular organizer of signalling competent membrane microdomains
and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion
and transmigration of human monocytes.
Journal of Biological Chemistry 04/2011; · 4.77 Impact Factor
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CancerSpectrum Knowledge Environment 11/2010; 102(22):1741; author reply 1741-2. · 14.07 Impact Factor
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Filomena Mazzei,
Simonetta Guarrera,
Alessandra Allione,
Valeria Simonelli,
Laura Narciso,
Flavia Barone,
Anna Minoprio,
Fulvio Ricceri, Ada Funaro,
Mariarosaria D'Errico,
Ulla Vogel,
Giuseppe Matullo,
Eugenia Dogliotti
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ABSTRACT: Several lines of evidence suggest an association between oxidative DNA-damage repair capacity and cancer risk. In particular, a DNA-glycosylase assay for removal of 8-oxoguanine (8-oxoG) in peripheral blood mononuclear cells (PBMC) has been successfully applied to identify populations with increased risk for lung cancer and squamous cell carcinomas of head and neck. In order to verify whether EBV-transformed lymphoblastoid cell lines (LCL) are a suitable surrogate for PBMC in specific DNA-repair phenotypic assays, a validation trial was conducted. PBMC from 20 healthy subjects were collected and an aliquot was transformed with EBV to obtain LCL. The ability of cell-free extracts from both cell types to incise a 3'-fluorescently labelled duplex oligonucleotide containing a single 8-oxoG (OGG assay) was evaluated. Since this activity is mediated predominantly by OGG1, the OGG1 gene expression was also measured. 8-oxoG DNA-glycosylase activity and OGG1 expression were significantly higher (p<0.0001) in LCL than in PBMC. However, while this assay was shown to be robust and reproducible when used on PBMC (intra-assay CV=8%), a high intra-culture variability was observed with LCL (intra-culture CV=16.8%). Neither differences on OGG1 gene expression nor the cell-cycle distribution seemed to account for this variability. Inter-individual variability of OGG activity in PBMC and LCL was not associated with OGG1 gene expression. We have therefore established a non-radioactive cleavage assay that can be easily applied to measure OGG activity in human PBMC. The use of LCL for DNA-repair genotype-phenotype correlation studies seems to be inappropriate, at least with cell-free based functional assays.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 10/2010; 718(1-2):62-7. · 2.85 Impact Factor
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Erika Ortolan,
Riccardo Arisio,
Simona Morone,
Paola Bovino,
Nicola Lo-Buono,
Giulia Nacci,
Rossella Parrotta,
Dionyssios Katsaros,
Ida Rapa,
Giuseppe Migliaretti,
Enza Ferrero,
Marco Volante, Ada Funaro
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ABSTRACT: CD157, an ADP-ribosyl cyclase-related cell surface molecule, regulates leukocyte diapedesis during inflammation. Because CD157 is expressed in mesothelial cells and diapedesis resembles tumor cell migration, we investigated the role of CD157 in ovarian carcinoma.
We assayed surgically obtained ovarian cancer and mesothelial cells and both native and engineered ovarian cancer cell lines for CD157 expression using flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR), and for adhesion to extracellular matrices, migration, and invasion using cell-based assays. We investigated invasion of human peritoneal mesothelial cells by serous ovarian cancer cells with a three-dimensional coculture model. Experiments were performed with or without CD157-blocking antibodies. CD157 expression in tissue sections from ovarian cancer patients (n = 88) was examined by immunohistochemistry, quantified by histological score (H score), and categorized as at or above or below the median value of 60, and compared with clinical parameters. Statistical tests were two-sided.
CD157 was expressed by ovarian cancer cells and mesothelium, and it potentiated the adhesion, migration, and invasion of serous ovarian cancer cells through different extracellular matrices. CD157-transfected ovarian cancer cells migrated twice as much as CD157-negative control cells (P = .001). Blockage of CD157 inhibited mesothelial invasion by serous ovarian cancer cells in a three-dimensional model. CD157 was expressed in 82 (93%) of the 88 epithelial ovarian cancer tissue specimens. In serous ovarian cancer, patients with CD157 H scores of 60 or greater had statistically significantly shorter disease-free survival and overall survival than patients with lower CD157 H scores (CD157 H score > or =60 vs <60: median disease-free survival = 18 months, 95% confidence interval [CI] = 5.92 to 30.07 vs unreached, P = .005; CD157 H score > or =60 vs <60: median overall survival = 45 months, 95% CI = 21.21 to 68.79 vs unreached, P = .024). Multivariable Cox regression showed that CD157 is an independent prognostic factor for recurrence (hazard ratio of disease recurrence = 3.01, 95% CI = 1.35 to 6.70, P = .007) and survival (hazard ratio of survival = 3.44, 95% CI = 1.27 to 9.31, P = .015).
CD157 plays a pivotal role in the control of ovarian cancer cell migration and peritoneal invasion, and it may be clinically useful as a prognostic tool and therapeutic target.
CancerSpectrum Knowledge Environment 08/2010; 102(15):1160-77. · 14.07 Impact Factor
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Andrea Zijno,
Paola Porcedda,
Francesca Saini,
Alessandra Allione,
Bruno Garofalo,
Francesca Marcon,
Simonetta Guarrera,
Valentina Turinetto,
Valentina Minieri, Ada Funaro,
Riccardo Crebelli,
Claudia Giachino,
Giuseppe Matullo
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ABSTRACT: As first task of a comprehensive investigation on DNA repair genotype-phenotype correlations, the suitability of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as surrogate of cryopreserved peripheral blood mononuclear cells (PBMCs) in DNA repair phenotypic assays was evaluated. To this aim the amount of DNA damage induced by gamma-rays and DNA repair capacity were evaluated in unstimulated (G(0)) and mitogen-simulated (G(2)) PBMC from 20 healthy subjects and in EBV-transformed LCL obtained from the same individuals. Phosphorylation of histone H2AX, micronuclei and chromosomal aberrations were the end-points investigated. The results obtained show higher basal frequencies of binucleated cells bearing micronuclei and nucleoplasmic bridge (NPB) in LCL with respect to PBMC, suggesting that EBV transformation may be associated with chromosomal instability. After irradiation, higher levels of micronuclei were induced in G(0)-treated PBMC compared to cycling LCL; conversely, NPB were more frequent in LCL than in PBMC. Moreover, higher levels of chromosomal aberrations were observed in G(2)-treated PBMC compared to LCL. Concerning gamma-H2AX measurements, phosphorylation levels 1h after treatment and dephosphorylation kinetics were basically similar in LCL and in PBMC. However, while Spearman's test showed a strong correlation between the results obtained in replicated experiments with PBMC, high inter-experimental variability and poor reproducibility was observed in the experiments performed with LCL, possibly due to the intrinsic instability of LCL. In summary, both the analysis of gamma-H2AX and the evaluation of chromosome damage highlighted a larger inter-experimental variability in the results obtained with LCL compared to PBMC. Noteworthy, the two set of results proved to lack any significant correlation at the individual level. These results indicate that LCL may be unsuitable for investigating genotype-phenotype correlations with phenotypic DNA repair assays, especially when low impact functional genetic variants are involved.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/2010; 684(1-2):98-105. · 2.85 Impact Factor
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ABSTRACT: CD157 is a glycosylphosphatidylinositol-anchored molecule encoded by a member of the CD38/ADP-ribosyl cyclase gene family, involved in the metabolism of NAD. Expressed mainly by cells of the myeloid lineage and by vascular endothelial cells, CD157 has a dual nature behaving both as an ectoenzyme and as a receptor. Although it lacks a cytoplasmic domain, and cannot transduce signals on its own, the molecule compensates for this structural limit by interacting with conventional receptors. Recent experimental evidence suggests that CD157 orchestrates critical functions of human neutrophils. Indeed, CD157-mediated signals promote cell polarization, regulate chemotaxis induced through the high affinity fMLP receptor and control transendothelial migration.
Frontiers in Bioscience 02/2009; 14:929-43. · 3.52 Impact Factor
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ABSTRACT: CD157 is a glycosyl phosphatidylinositol-anchored cell surface glycoprotein prevalently expressed by myeloid and bone marrow stromal cells. Functional analysis of CD157 by means of agonistic monoclonal antibodies demonstrated that CD157 ligation on neutrophils regulates Ca2+ homeostasis and promotes polarization, thus controlling their adhesive and migratory properties. Here we show that on monocytes (i) CD157 localizes in GM1-enriched lipid rafts, (ii) is involved in adhesion to extracellular matrix proteins, (iii) controls MCP-1-induced chemotaxis, (iv) regulates transendothelial migration, and (v) exerts receptor functions. CD157 lacks a cytoplasmic domain, so, it must associate with membrane spanning receptors to transduce signals. On monocytes, CD157 establishes structural and functional cross-talk with both β1-integrin and β2-integrin. Indeed, co-capping experiments showed that CD157 is spatially associated with β1- and β2-integrin complexes: ligand-induced clustering of both integrin complexes causes co-localization of CD157. The association was confirmed at structural level since immunoprecipitation of both β1 and β2-integrin induced co-precipitation of CD157. In the THP-1 myelomonocytic line CD157 cross-linking promotes a signaling cascade which involves phosphorylation of AKT-1 and p42/44 (ERK1/2). Simultaneous ligation of CD157 and β1 and β2-integrin leads to increased AKT-1 and ERK1/2 phosphorylation at all time intervals considered, indicating the existence of functional interaction between CD157, β1 and β2-integrins. These findings demonstrate that CD157 is a component of a multimolecular complex orchestrating monocyte adhesion and migration.
Cytokine. 01/2009; 48:130.
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Ada Funaro,
Giorgio Gribaudo,
Anna Luganini,
Erika Ortolan,
Nicola Lo Buono,
Elisa Vicenzi,
Luca Cassetta,
Santo Landolfo,
Richard Buick,
Luca Falciola,
Marianne Murphy,
Gianni Garotta,
Fabio Malavasi
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ABSTRACT: Human monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective.Human cytomegalovirus (HCMV) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. The available therapeutic armamentarium (e.g. HCMV hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mAb may be a decisive alternative in the prevention of primary and re-activated HCMV infections in these patients.
The purpose of this study was to generate neutralizing mAb against HCMV from the immunological repertoire of immune donors. To this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human B-lymphocytes are stimulated with TLR9-agonists and IL-2; second, after both additives are removed, the cells are infected with EBV. Using this strategy we obtained 29 clones secreting IgG neutralizing the HCMV infectivity; four among these were further characterized. All of the mAbs neutralize the infection in different combinations of HCMV strains and target cells, with a potency approximately 20 fold higher than that of the HCMV hyperimmune globulins, currently used in transplant recipients. Recombinant human monoclonal IgG1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated.
The technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mAbs for immunotherapeutic uses.
BMC Biotechnology 12/2008; 8:85. · 2.35 Impact Factor
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ABSTRACT: The membrane proteins CD38 and CD157 belong to an evolutionarily conserved family of enzymes that play crucial roles in human physiology. Expressed in distinct patterns in most tissues, CD38 (and CD157) cleaves NAD(+) and NADP(+), generating cyclic ADP ribose (cADPR), NAADP, and ADPR. These reaction products are essential for the regulation of intracellular Ca(2+), the most ancient and universal cell signaling system. The entire family of enzymes controls complex processes, including egg fertilization, cell activation and proliferation, muscle contraction, hormone secretion, and immune responses. Over the course of evolution, the molecules have developed the ability to interact laterally and frontally with other surface proteins and have acquired receptor-like features. As detailed in this review, the loss of CD38 function is associated with impaired immune responses, metabolic disturbances, and behavioral modifications in mice. CD38 is a powerful disease marker for human leukemias and myelomas, is directly involved in the pathogenesis and outcome of human immunodeficiency virus infection and chronic lymphocytic leukemia, and controls insulin release and the development of diabetes. Here, the data concerning diseases are examined in view of potential clinical applications in diagnosis, prognosis, and therapy. The concluding remarks try to frame all of the currently available information within a unified working model that takes into account both the enzymatic and receptorial functions of the molecules.
Physiological Reviews 08/2008; 88(3):841-86. · 26.87 Impact Factor
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ABSTRACT: Mutation detection remains problematic for large genes, primarily because PCR-based methodology fails to detect heterozygous deletions and any duplication. In the ATM gene only a handful of multi-exon deletions have been described to date, and this type of mutation has been considered rare. To address this issue we tested a new MLPA (Multiplex Ligation Probe Amplification) kit that covers 33 of the 66 ATM exons, using for controls two previously characterized genomic deletions in addition to three A-T patients, taken from a survey of nine, who had missing four mutations unidentified after conventional mutation screening. We identified for the first time: 1) a approximately 41 kb genomic duplication spanning exons 4-20 (c.-30_2816dup41kb)(a.k.a., ATM dup 41 kb); 2) a novel genomic deletion including exon 31, and 3) in hemizygosis a point mutation in the non-deleted exon 31. In this study we extended mutation detection to nine new Italian A-T patients, using a combined approach of haplotype analysis, DHPLC and MLPA. Overall we achieved a mutation detection rate of >97%, and can now define a spectrum of ATM mutations based on twenty-one consecutive Italian families with A-T.
Annals of Human Genetics 02/2008; 72(Pt 1):10-8. · 2.57 Impact Factor
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Sonia Molina,
Valérie Castet,
Chantal Fournier-Wirth,
Lydiane Pichard-Garcia,
Rachel Avner,
Dror Harats,
Joseph Roitelman,
Ronald Barbaras,
Pierre Graber,
Paola Ghersa,
Moshe Smolarsky, Ada Funaro,
Fabio Malavasi,
Dominique Larrey,
Joliette Coste,
Jean-Michel Fabre,
Antonio Sa-Cunha,
Patrick Maurel
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ABSTRACT: The direct implication of low-density lipoprotein receptor (LDLR) in hepatitis C virus (HCV) infection of human hepatocyte has not been demonstrated. Normal primary human hepatocytes infected by serum HCV were used to document this point.
Expression and activity of LDLR were assessed by RT-PCR and LDL entry, in the absence or presence of squalestatin or 25-hydroxycholesterol that up- or down-regulates LDLR expression, respectively. Infection was performed in the absence or presence of LDL, HDL, recombinant soluble LDLR peptides encompassing full-length (r-shLDLR4-292) or truncated (r-shLDLR4-166) LDL-binding domain, monoclonal antibodies against r-shLDLR4-292, squalestatin or 25-hydroxycholesterol. Intracellular amounts of replicative and genomic HCV RNA strands used as end point of infection were assessed by RT-PCR.
r-shLDLR4-292, antibodies against r-shLDLR4-292 and LDL inhibited viral RNA accumulation, irrespective of genotype, viral load or liver donor. Inhibition was greatest when r-shLDLR4-292 was present at the time of inoculation and gradually decreased as the delay between inoculation and r-shLDLR4-292 treatment increased. In hepatocytes pre-treated with squalestatin or 25-hydroxycholesterol before infection, viral RNA accumulation increased or decreased in parallel with LDLR mRNA expression and LDL entry.
LDLR is involved at an early stage in infection of normal human hepatocytes by serum-derived HCV virions.
Journal of Hepatology 04/2007; 46(3):411-9. · 9.26 Impact Factor
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ABSTRACT: Paracellular diapedesis, a key step in leukocyte recruitment to the site of inflammation, occurs at endothelial junctions and is regulated by highly coordinated interactions between leukocytes and endothelium. We found that CD157, a glycosylphosphatidylinositol-anchored ectoenzyme belonging to the NADase/ADP-ribosyl cyclase family, plays a crucial role for neutrophil diapedesis, because its ligation with specific monoclonal antibodies (both on neutrophils or endothelial cells) results in altered neutrophil movement on the apical surface of endothelium and, ultimately, in loss of diapedesis. Real-time microscopy revealed that CD157 behaves as a sort of compass during the interaction between neutrophils and endothelial cells; indeed, following CD157 ligation, neutrophils appear disoriented, meandering toward junctions where they eventually stop without transmigrating. These findings are relevant in vivo because CD157-deficient neutrophils obtained from patients with paroxysmal nocturnal hemoglobinuria are characterized by a severely impaired diapedesis.
Blood 01/2007; 108(13):4214-22. · 9.90 Impact Factor
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ABSTRACT: In patients affected by Ataxia-Telangiectasia (A-T), mutations in the ATM gene lead to loss-of-function alleles. Nonsense, splice-site variants, small insertions or deletions (frameshifts) and missense are the most commonly found mutations. Large genomic deletions (LGDs) are rare (approximately 1%) but can lead to the same phenotype. In compound heterozygotes, deletions are not detected by most screening strategies. We analysed the ATM gene in 12 unrelated Italian A-T patients and identified all 24 mutated alleles. Twelve mutations were novel. Standardized SNP and STR haplotyping followed by DHPLC screening of genomic DNA, allowed all but three mutations to be detected (approximately 87.5%). The remaining mutations required RT-PCR analysis of ATM transcript and Southern blotting of genomic DNA. We found three LGDs: one of 8.5 and two identical of 18 kb spanning exons 32-36 and 21-29, respectively. The breakpoints of these deletions were sequenced in an attempt to understand the mechanisms of mutations; both deletions involved regions rich in repeated elements.
Human Mutation 11/2006; 27(10):1061. · 5.69 Impact Factor
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Silvia Svegliati Baroni,
Mariarosaria Santillo,
Federica Bevilacqua,
Michele Luchetti,
Tatiana Spadoni,
Matteo Mancini,
Paolo Fraticelli,
Paola Sambo, Ada Funaro,
Andrius Kazlauskas,
Enrico V Avvedimento,
Armando Gabrielli
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ABSTRACT: Systemic sclerosis (scleroderma) is characterized by immunologic abnormalities, injury of endothelial cells, and tissue fibrosis. Abnormal oxidative stress has been documented in scleroderma and linked to fibroblast activation. Since platelet-derived growth factor (PDGF) stimulates the production of reactive oxygen species (ROS) and since IgG from patients with scleroderma reacts with human fibroblasts, we tested the hypothesis that patients with scleroderma have serum autoantibodies that stimulate the PDGF receptor (PDGFR), activating collagen-gene expression.
We analyzed serum from 46 patients with scleroderma and 75 controls, including patients with other autoimmune diseases, for stimulatory autoantibodies to PDGFR by measuring the production of ROS produced by the incubation of purified IgG with mouse-embryo fibroblasts carrying inactive copies of PDGFR alpha or beta chains or the same cells expressing PDGFR alpha or beta. Generation of ROS was assayed with and without specific PDGFR inhibitors. Antibodies were characterized by immunoprecipitation, immunoblotting, and absorption experiments.
Stimulatory antibodies to the PDGFR were found in all the patients with scleroderma. The antibodies recognized native PDGFR, inducing tyrosine phosphorylation and ROS accumulation. Autoantibody activity was abolished by preincubation with cells expressing the PDGFR alpha chain or with recombinant PDGFR or by PDGFR tyrosine kinase inhibitors. Stimulatory PDGFR antibodies selectively induced the Ha-Ras-ERK1/2 and ROS cascades and stimulated type I collagen-gene expression and myofibroblast phenotype conversion in normal human primary fibroblasts.
Stimulatory autoantibodies against PDGFR appear to be a specific hallmark of scleroderma. Their biologic activity on fibroblasts strongly suggests that they have a causal role in the pathogenesis of the disease.
New England Journal of Medicine 07/2006; 354(25):2667-76. · 53.30 Impact Factor
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ABSTRACT: CD157, a glycosylphosphatidylinositol (GPI)-anchored protein encoded by a member of the CD38 NADase/ADP-ribosyl cyclase gene family, is expressed on the surface of most human circulating neutrophils. This work demonstrates that CD157 is a receptor that induces reorganization of the cytoskeleton and significant changes in cell shape, and that signals mediated by CD157 act through modulation of cytosolic Ca(2+) concentration. These signals are independent of the products of CD157's enzymatic activities (ie, cyclic adenosine diphosphate [ADP]-ribose and ADP-ribose). Indeed, the enzymatic activities of CD157 in circulating neutrophils as well as in dimethyl sulfoxide (DMSO)-differentiated (CD157(+)/CD38(-)) HL-60 cells, are hardly detectable. This work also shows that the receptorial activity relies on cross-talk between CD157 and beta(2) integrin. CD157 localizes in GM1-enriched lipid rafts and, upon activation, it migrates to the uropod, a structure specialized in motility and adhesive functions. Indeed, CD157 is involved in adhesion to extracellular matrix proteins and in chemotaxis induced in vitro by formyl-methionyl-leucyl-phenylalanine (fMLP). These findings were consistent with the results obtained in neutrophils from patients with paroxysmal nocturnal hemoglobinuria (PNH), in which CD157 is deficient. These neutrophils showed constant defects in adhesion and migration. Our data attribute specific and crucial roles to CD157 in the regulation of innate immunity during inflammation.
Blood 01/2005; 104(13):4269-78. · 9.90 Impact Factor
