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1.
Rhinology ; 43(4): 251-6, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16405267

ABSTRACT

Specific ultrastructural findings have widely been described in case of obstructive nasal diseases due to congenital defects. Ciliary impairment has in particular been observed as the main pathological feature in these conditions. In this study, nasal mucosal samples from different pathologies have been collected via the "brushing" technique and analysed by transmission electron microscopy. TEM analysis was focused on specific features, such as the numerical array of peripheral and central doublets of the cilium axoneme, including eventual microtubular disarrangement; partial or total loss of inner and/or outer dynein arms; defects of radial spokes and nexin links; disorientation of the ciliary axis in closely adjacent cilia, calculating the angle between the line crossing the central microtubular core and the horizontal ciliary axis and compound cilia (CC). Statistical comparison was carried out between study and control groups. A significant incidence of organic ciliary defects was found not only in patients with inflammatory processes, but mostly in those supposed to have a long-lasting nasal respiratory disease due to mechanical stenosis in relation to septum deviation and turbinate hypertrophy. Prevalence and percentage of compound cilia were instead more related to inflammatory conditions. The "brushing" technique can be considered an easy and reliable method for the assessment of the condition of the nasal mucosa. According to the findings derived from this study, mechanical nasal obstruction seems to cause major alterations on the nasal ciliary arrangement, thus determining a functional impairment on the whole nasal function.


Subject(s)
Cilia/ultrastructure , Ciliary Motility Disorders/pathology , Nasal Obstruction/pathology , Adult , Analysis of Variance , Chi-Square Distribution , Female , Humans , Male , Middle Aged
3.
J Invest Dermatol ; 116(4): 623-8, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11286634

ABSTRACT

Fibroblast growth factor 10 is a novel member of the fibroblast growth factor family, which is involved in morphogenesis and epithelial proliferation. It is highly homologous to the keratinocyte growth factor (or fibroblast growth factor 7), a key mediator of keratinocyte growth and differentiation. Both fibroblast growth factor 10 and keratinocyte growth factor bind with high affinity to the tyrosine kinase keratinocyte growth factor receptor. Here we analyzed the effect of fibroblast growth factor 10 on primary cultures of human keratinocytes, grown in chemically defined medium, and we compared the proliferative and differentiative cell responses to fibroblast growth factor 10 with those induced by keratinocyte growth factor and epidermal growth factor. Cell counting, 5-bromo-2'-deoxyuridine incorporation, and western blot analysis showed that fibroblast growth factor 10, similarly to keratinocyte growth factor, not only is a potent mitogen for human keratinocytes, but also promotes the expression of both early differentiation markers K1 and K10 and late differentiation marker filaggrin in response to the Ca2+ signal, and seems to sustain the proliferative activity in suprabasal stratified cells. Immunoprecipitation/western blot analysis revealed that fibroblast growth factor 10, similarly to keratinocyte growth factor, is able to induce tyrosine phosphorylation of keratinocyte growth factor receptor and of cellular substrates such as PLCgamma.


Subject(s)
Fibroblast Growth Factors/pharmacology , Keratinocytes/cytology , Keratinocytes/drug effects , Receptors, Fibroblast Growth Factor , Antigens, Differentiation/metabolism , Cell Differentiation , Cell Division , Cells, Cultured , Epidermal Growth Factor/pharmacology , Fibroblast Growth Factor 10 , Fibroblast Growth Factor 7 , Fibroblast Growth Factors/metabolism , Filaggrin Proteins , Humans , Intermediate Filament Proteins/metabolism , Phosphorylation , Receptor, Fibroblast Growth Factor, Type 2 , Receptors, Growth Factor/metabolism , Tyrosine/metabolism
4.
Cell Growth Differ ; 11(11): 607-14, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11095249

ABSTRACT

Keratinocyte growth factor (KGF) is involved in the control of proliferation and differentiation of human keratinocytes. It binds to, and activates, the tyrosine kinase KGF receptor (KGFR), a splicing transcript variant of the fibroblast growth factor receptor 2. We have previously shown (C. Marchese et al., Cell Growth Differ., 8: 989-997, 1997) that differentiation of primary cultured keratinocytes triggered by high Ca2+ concentrations in the growing medium induced up-regulation of KGFR expression, which suggested that KGFR may play a crucial role in the control of the proliferative/differentiative program during transition from basal to suprabasal cells. Here we analyzed the process of modulation of the expression of KGFRs in the human keratinocyte cell line HaCaT, widely used as a model to study keratinocyte differentiation. Western blot and double immunofluorescence for KGFR and the K1 differentiation marker showed that cell differentiation and stratification induced by confluence and high cell density correlated with an increase in KGFR expression. KGFRs, present on suprabasal differentiated cells, appeared to be efficiently tyrosine-phosphorylated by KGF, which indicated that the receptors up-regulated by differentiation can be functionally activated by ligand binding. Bromodeoxyuridine incorporation assay revealed that a significant portion of suprabasal differentiated cells expressing KGFR seemed to be still able to synthesize DNA and to proliferate in response to KGF, which suggested that increased KGFR expression may be required for retention of the proliferative activity.


Subject(s)
Fibroblast Growth Factors , Keratinocytes/cytology , Keratinocytes/metabolism , Receptors, Fibroblast Growth Factor , Receptors, Growth Factor/metabolism , Up-Regulation , Blotting, Western , Bromodeoxyuridine/metabolism , Cell Count , Cell Differentiation/drug effects , Cell Division/drug effects , Cell Line , Fibroblast Growth Factor 10 , Fibroblast Growth Factor 7 , Fluorescent Antibody Technique , Growth Substances/pharmacology , Humans , Keratinocytes/drug effects , Keratinocytes/ultrastructure , Microscopy, Electron , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Receptor, Fibroblast Growth Factor, Type 2 , Receptors, Growth Factor/genetics , Recombinant Fusion Proteins , Up-Regulation/drug effects
5.
Am J Pathol ; 156(4): 1417-24, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10751365

ABSTRACT

Investigation of ErbB2 immunity in human breast cancer employing recombinant expression sources in immunoblot analysis revealed ErbB2-specific antibodies of the IgG isotype in sera of 14 of 71 cancer patients and 1 of 31 normal donors. Reactivity was confirmed on ErbB2-specific immunoprecipitates. Independent evidence of existing ErbB2 immunity was obtained after in vitro transformation of peripheral blood leukocytes from six positive patients. Furthermore, in vitro immortalization of B-lymphocytes unmasked existent ErbB2 immunity in 1 of 8 patients negative for ErbB2 serum antibodies. Determining shed ErbB2 extracellular domain as an indirect measure of tumor burden in ErbB2-positive malignancy, elevated serum levels were observed in 16 of 71 breast cancer and 1 of 31 normal donor sera. Strikingly, existing ErbB2 immunity correlated significantly with elevated shed ErbB2 ectodomain among the patients analyzed. Incidence of both ErbB2 immunity and elevated ErbB2 extracellular domain increased with a progressed disease stage and was significantly associated with metastatic breast cancer. These observations implicate soluble ErbB2 amounts in vivo in the development of ErbB2 immunity in breast cancer. They further project serum analysis of ErbB2 immunity and soluble ectodomain as potential markers of disease progression in ErbB2-positive malignancy.


Subject(s)
Breast Neoplasms/immunology , Receptor, ErbB-2/immunology , 3T3 Cells , Animals , Antibodies/blood , Antibody Formation , B-Lymphocytes/metabolism , Breast Neoplasms/blood , Breast Neoplasms/physiopathology , Cell Line, Transformed , Female , Humans , Mice , Protein Structure, Tertiary , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Solubility
6.
Histochem Cell Biol ; 113(1): 43-50, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10664069

ABSTRACT

The expression and surface distribution of monosialoganglioside GM3 on the plasma membranes of NIH3T3 fibroblasts cultured at semiconfluence were analyzed by immunofluorescence as well as by immunogold electron microscopy on thin sections and surface replicas. The GM3 expression was highly variable from cell to cell and the distribution of the ganglioside on the positive cells appeared punctate. Quantitative immunogold electron microscopy showed the existence of well-defined GM3 clusters of different sizes scattered all over the cell surfaces. Double immunofluorescence analysis of 5-bromo-2'-deoxyuridine incorporation to identify proliferating cells and of GM3 expression indicated that most of the GM3-positive cells appear unable to synthesize DNA and demonstrated a growth-dependent expression of GM3.


Subject(s)
3T3 Cells/cytology , 3T3 Cells/metabolism , G(M3) Ganglioside/biosynthesis , G(M3) Ganglioside/physiology , Growth Inhibitors/physiology , 3T3 Cells/ultrastructure , Animals , Cell Division/physiology , Cell Membrane/metabolism , Fluorescent Antibody Technique, Indirect , Mice , Microscopy, Confocal , Microscopy, Immunoelectron
7.
FEBS Lett ; 465(1): 39-46, 2000 Jan 07.
Article in English | MEDLINE | ID: mdl-10620703

ABSTRACT

We report the molecular characterization of five human monoclonal antibodies, BAB1-5 (BAB1: IgG(1); BAB4: IgG(2); BAB2, 3, 5: IgG(4)), with specificity for the major birch pollen allergen, Bet v 1. BAB1-5 were obtained after immunotherapy and contained a high degree of somatic mutations indicative of an antigen-driven affinity maturation process. While BAB1 inhibited the binding of patients IgE to Bet v 1, BAB2 increased IgE recognition of Bet v 1, and, even as Escherichia coli-expressed Fab, augmented Bet v 1-induced immediate type skin reactions. The demonstration that IgG antibodies can enhance allergen-induced allergic reactions is likely to explain the unpredictability of specific immunotherapy.


Subject(s)
Allergens/immunology , Antibodies, Monoclonal/chemistry , Hypersensitivity, Immediate/immunology , Plant Proteins/immunology , Pollen/immunology , Amino Acid Sequence , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Antibody Specificity , Antigens, Plant , Epitopes/immunology , Escherichia coli/metabolism , Humans , Hypersensitivity, Immediate/therapy , Immunoglobulin E/immunology , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/immunology , Molecular Sequence Data , Plant Proteins/chemistry
8.
Int J Oncol ; 15(3): 431-5, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10427121

ABSTRACT

Immunohistochemical analysis of the expression of keratinocyte growth factor receptor (KGFR) was performed in human endometrial carcinomas from 18 patients and in normal proliferative and secretory endometrium. The level of immunostaining was correlated with the clinico-pathological characteristics of the endometrial carcinoma patients and with the parallel expression of the epidermal growth factor receptor (EGFR) and erbB-2. The results showed that KGFR expression increased with the stage of the tumor and that the simultaneous overexpression of the three growth factor receptors appeared to be related to the depth of myometrial invasion. Taken together, these observations suggest that KGFR may represent an additional prognostic indicator in endometrial cancer.


Subject(s)
Adenocarcinoma/chemistry , Endometrial Neoplasms/chemistry , Neoplasm Proteins/analysis , Receptors, Fibroblast Growth Factor , Adult , Aged , Disease Progression , ErbB Receptors/analysis , Female , Humans , Immunohistochemistry , Keratinocytes , Middle Aged , Neoplasm Invasiveness , Prognosis , Receptor, ErbB-2/analysis , Receptor, Fibroblast Growth Factor, Type 2 , Receptors, Growth Factor/analysis
9.
Int Arch Allergy Immunol ; 118(2-4): 190-2, 1999.
Article in English | MEDLINE | ID: mdl-10224374

ABSTRACT

BACKGROUND: Antigen recognition by antibodies of different isotypes can result in completely different effects as exemplified by Type I allergy. While the IgE-antibody-mediated release of biological mediators constitutes the immunopathological basis for the immediate symptoms observed in allergic patients, allergen-specific IgG antibodies are thought to have protective effects. METHODS: Cell lines secreting five human monoclonal IgG antibodies (BAB1-BAB5) with specificity for the major birch pollen allergen Bet v 1 were established from a birch-pollen-allergic patient who had received birch- pollen-specific immunotherapy. The influence of the Bet v 1-specific IgG antibodies on IgE binding to Bet v 1 was investigated. BAB2 was expressed in Escherichia coli as recombinant Fab, purified and tested for its ability to modulate Bet v 1-induced immediate-type skin reactions. RESULTS: The BAB antibodies belonged to different IgG subclasses (BAB1: IgG1; BAB2, BAB3, BAB5: IgG4; and BAB4: IgG2) reflecting a tendency towards Th2. BAB1 represented the only antibody which strongly blocked IgE binding to Bet v 1, whereas BAB 3-BAB5 had little effect on IgE binding. Surprisingly, natural BAB2 antibodies as well as recombinant BAB2 Fabs strongly enhanced IgE binding to Bet v 1 and Bet v 1-induced immediate-type skin reactions and thus represent 'enhancing antibodies'. CONCLUSION: The demonstration that anti-allergen IgG antibodies can also enhance IgE binding to a given allergen explains the unpredictability of specific immunotherapy as well as the controversy on the role of IgG in atopy.


Subject(s)
Allergens/immunology , Immunoglobulin Fab Fragments/immunology , Immunoglobulin G/immunology , Plant Proteins/immunology , Antibody Specificity , Antigens, Plant , Epitopes , Escherichia coli , Genes, Immunoglobulin , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin G/genetics , Pollen , Recombinant Proteins/genetics , Recombinant Proteins/immunology
10.
Oncogene ; 18(6): 1267-75, 1999 Feb 11.
Article in English | MEDLINE | ID: mdl-10022808

ABSTRACT

Employing NIH3T3 transfectants with individual human ErbB receptor coding sequences as recombinant antigen sources, we detected by immunoblot analysis specific immunoreactivity against all four ErbB receptors among 13 of 41 sera obtained from patients with different types of epithelial malignancies. Overall, serum positivity was most frequently directed against ErbB2 followed by EGFR, ErbB3 and ErbB4. Specificity patterns comprised tumor patients with unique serum reactivity against ErbB2 or ErbB4. Moreover, approximately half of the positive sera exhibited concomitant reactivity with multiple ErbB receptors including EGFR and ErbB2, EGFR and ErbB4, ErbB2 and ErbB3 or EGFR, ErbB2 and ErbB3. Serum reactivity was confirmed for the respective ErbB receptors expressed by human tumor cells and corroborated on receptor-specific immunoprecipitates. Positive sera contained ErbB-specific antibodies of the IgG isotype. Representative immunohistochemical analysis of tumor tissues suggested overexpression of ErbB receptors for which serum antibodies were detectable in five of six patients. These findings implicate multiple ErbB receptors including ErbB3 and ErbB4 in addition to EGFR and ErbB2 in primary human cancer. Heterogeneity of natural ErbB-specific responses in cancer patients warrants their evaluation in light of immunotherapeutic approaches targeting these receptors.


Subject(s)
Antibodies, Neoplasm/blood , ErbB Receptors/immunology , Proto-Oncogene Proteins/immunology , Receptor, ErbB-2/immunology , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Carcinoma/immunology , ErbB Receptors/isolation & purification , Humans , Immunoglobulin Isotypes , Immunohistochemistry , Lymphoma/immunology , Neoplasms, Glandular and Epithelial/immunology , Proto-Oncogene Proteins/isolation & purification , Receptor, ErbB-2/isolation & purification , Receptor, ErbB-3 , Receptor, ErbB-4 , Recombinant Proteins/immunology
11.
J Allergy Clin Immunol ; 99(3): 374-84, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9058694

ABSTRACT

BACKGROUND: Bet v 1 and homologous proteins represent major allergens for almost 95% of patients allergic to tree pollen and approximately 70% of those allergic to fruits and vegetables. As yet, no continuous (sequential) IgE epitopes have been determined for Bet v 1, and evidence has accumulated that Bet v 1 IgE epitopes belong to the conformational (discontinuous) type. OBJECTIVE: A panel of 85 mouse monoclonal anti-Bet v 1 antibodies was raised as a tool with which to study the interaction of human IgE antibodies with Bet v 1. METHODS: The epitopes of selected monoclonal antibodies (mAbs) were characterized by mapping with synthetic overlapping peptides and by cross-competition experiments. Cross-reactivity of Bet v 1-specific mAbs with tree and plant food allergens was investigated by Western blotting. The influence of Bet v 1-specific mAbs on the IgE-Bet v 1 interaction was studied by competition assays with immobilized purified recombinant Bet v 1 and by basophil histamine release experiments. RESULTS: Antibodies that increased the IgE binding to Bet v 1 up to fivefold could be defined, whereas others inhibited IgE binding to Bet v 1 up to 99% and competed with the Bet v 1-induced histamine release from patients' basophils. CONCLUSION: The activity of the enhancing antibodies is interpreted as a stabilization of Bet v 1 states/IgE epitopes, which are either more accessible for certain IgE antibodies or are recognized with higher affinity. Those mAbs that competed with the Bet v 1-IgE interaction, if humanized or produced as recombinant antibody fragments, might be considered as potential tools for local allergy therapy.


Subject(s)
Allergens , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Epitopes/analysis , Immunoglobulin E/immunology , Plant Proteins/immunology , Animals , Antibodies, Blocking/immunology , Antibodies, Monoclonal/isolation & purification , Antigens, Plant , Basophils/immunology , Basophils/metabolism , Blotting, Western , Chromatography, Affinity , Cross Reactions/immunology , Enzyme-Linked Immunosorbent Assay , Histamine Release , Humans , Mice , Mice, Inbred BALB C , Peptide Mapping , Plant Proteins/genetics , Precipitin Tests , Recombinant Proteins/immunology
12.
J Immunol ; 157(2): 956-62, 1996 Jul 15.
Article in English | MEDLINE | ID: mdl-8752951

ABSTRACT

Birch pollen allergy is a very frequent pathology in Europe and North America. More than 95% of the tree pollen allergic patients display IgE reactivity against Bet v 1, the major birch pollen allergen. Starting with PBL from a patient desensitized by immunotherapy, we have generated five B cell lines (BAB1 to BAB5) that secrete human IgG mAbs of high affinity for Bet v 1. Although competition studies indicated that these human IgG mAb recognized different epitopes, broad cross-reactivity was found with Bet v 1 homologous allergens present in tree pollens and plant-derived foods. When tested for interference with allergic patients' IgE, BAB1 inhibited (by 80-100%) the binding of IgE to nitrocellulose-blotted Bet v 1, while BAB2 enhanced it. The biologic significance of the ability of BAB1 to interfere with patients' IgE binding is indicated by the finding that BAB1 completely inhibited Bet v 1-induced histamine release from allergic patients' basophils. Allergen-specific human IgG mAbs such as BAB1, which presents high blocking activity in both immunochemical and cellular IgE competition experiments, might have therapeutical application.


Subject(s)
Adjuvants, Immunologic/pharmacology , Allergens/metabolism , Antibodies, Monoclonal/pharmacology , Binding Sites, Antibody/drug effects , Immunoglobulin E/metabolism , Immunoglobulin G/pharmacology , Plant Proteins/metabolism , Pollen/immunology , Adjuvants, Immunologic/biosynthesis , Adjuvants, Immunologic/chemistry , Allergens/drug effects , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Antibody Specificity , Antigens, Plant , Basophils/immunology , Binding, Competitive , Cross Reactions , Epitope Mapping , Histamine Release/drug effects , Humans , Immunoglobulin E/drug effects , Immunoglobulin G/biosynthesis , Immunoglobulin G/chemistry , Plant Proteins/drug effects
13.
Int J Oncol ; 8(6): 1127-35, 1996 Jun.
Article in English | MEDLINE | ID: mdl-21544473

ABSTRACT

We report a novel anti-CEA monoclonal antibody (MAb) designated R4, which mediates antibody-dependent cell-mediated cytotoxicity (ADCC) of human colon carcinoma cells and displays differential reactivity for human carcinomas versus the normal counterparts. R4 (IgG1) reacted with the cell surface of 6 colon carcinoma cell lines expressing CEA. Western blot analysis and epitope mapping using native and baculovirus recombinant CEA and non specific cross-reacting antigen (NCA) demonstrated that MAb R4 recognizes a proteinic epitope located on the 3' end of the domain I shared by CEA and NCA molecules. Immunohistochemical analysis demonstrated an intense staining of MAb R4 with the majority of the neoplastic tissues tested, including colon (13/13), stomach (2/2), breast (9/10), lung (7/10) and endometrial (2/4) carcinomas, whereas no reactivity with the correspondent normal tissues was observed. Using human PBLs from healthy donors as effector cells, we have shown that MAb R4 mediated antibody dependent-cell mediated cytotoxicity (ADCC) of human carcinoma cells LS-174T, CBS and WiDr. This activity was enhanced after PBLs activation with interleukin-2 (IL-2). The specificity of MAb R4 for an epitope shared by two tumor overexpressed antigens, CEA and NCA, resulting in an intense reactivity with neoplastic cells and the peculiar property to mediate ADCC, indicate that MAb R4 might be a novel powerful reagent for diagnostic and immunotherapy of carcinoma patients.

14.
Int J Oncol ; 8(6): 1119-26, 1996 Jun.
Article in English | MEDLINE | ID: mdl-21544472

ABSTRACT

In this study the immunohistochemical expressions of epidermal growth factor receptor (EGF-R) and erbB-2 in endometrial carcinomas at different stages of disease progression were evaluated and correlated with clinicopathological prognostic variables. Our results indicate that EGF-R and erbB-2 molecules are expressed in normal endometrium as well as in the majority of the carcinomas evaluated (83% and 92% respectively). The intensity of the immunostaining varied greatly (1+ to 3+) in the different tumors. Within these tumors we focused on those characterized by high levels of expression (i.e. overexpression). Expression and overexpression of EGF-R has been associated with poorly differentiated tumors and with a 3.3 times higher risk of a more rapid disease relapse. Overexpression of the erbB-2 oncoprotein was significatively correlated with depth of myometrial invasion (M0-M1 vs M2-M3 p=0.05), pathological stage (stage I vs II-IV p=0.02) and grade of tumor differentiation (G1 vs G2-G3 p=0.001). In particular, c-erbB-2 overexpression was able to discriminate between the different subsets of stage I carcinomas. None of the IA tumors overexpressed the oncoprotein, as compared to IB and IC (41% and 100% respectively, p=0.04). This finding highlights a role for erbB-2 protein as a prognostic parameter in stage I endometrial carcinoma patients.

15.
Ann Otolaryngol Chir Cervicofac ; 105(2): 131-3, 1988.
Article in French | MEDLINE | ID: mdl-3259413

ABSTRACT

The authors discuss their double-blind experiments on 40 allergic rhinitis affected patients, aged 15 to 50 years. Twenty were treated with H2 antagonists (Cimetidine) and twenty with placebo. The clinical assessment of the effectiveness of the treatment was carried out on a range of subjective and objective parameters taken before and after treatment. The IgE, IgA, IgG and IgM serum rate was measured. An improvement in the symptomatology was noted in 15 of the 20 Cimetidine treated patients and none in the placebo group. The results have shown a significant percentage decrease in the total serum IgE values after treatment, compared to initial ones, while no significant change was observed in the values of IgA, IgG and IgM. This decrease in IgE is thus a specific class. As it has been demonstrated that a subpopulation of lymphocyte T suppressors acts selectively on IgE producing B cells, the authors believe that only on these elements, carriers of H2 membrane receptors, can Cimetidine act to induce a decrease in total serum IgE levels and therefore a reduction in degranulation processes.


Subject(s)
Immunoglobulins/metabolism , Rhinitis, Allergic, Perennial/blood , Adolescent , Adult , B-Lymphocytes/physiology , Cimetidine/pharmacology , Humans , Immunoglobulin E/analysis , Middle Aged , Receptors, Histamine H2/drug effects , Rhinitis, Allergic, Perennial/drug therapy , Skin Tests , T-Lymphocytes/physiology
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