Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 28
Filter
Add more filters

Publication year range
1.
Int J Mol Sci ; 21(21)2020 Nov 02.
Article in English | MEDLINE | ID: mdl-33147779

ABSTRACT

Hyaline fibromatosis syndrome (HFS), resulting from ANTXR2 mutations, is an ultra-rare disease that causes intestinal lymphangiectasia and protein-losing enteropathy (PLE). The mechanisms leading to the gastrointestinal phenotype in these patients are not well defined. We present two patients with congenital diarrhea, severe PLE and unique clinical features resulting from deleterious ANTXR2 mutations. Intestinal organoids were generated from one of the patients, along with CRISPR-Cas9 ANTXR2 knockout, and compared with organoids from two healthy controls. The ANTXR2-deficient organoids displayed normal growth and polarity, compared to controls. Using an anthrax-toxin assay we showed that the c.155C>T mutation causes loss-of-function of ANTXR2 protein. An intrinsic defect of monolayer formation in patient-derived or ANTXR2KO organoids was not apparent, suggesting normal epithelial function. However, electron microscopy and second harmonic generation imaging showed abnormal collagen deposition in duodenal samples of these patients. Specifically, collagen VI, which is known to bind ANTXR2, was highly expressed in the duodenum of these patients. In conclusion, despite resistance to anthrax-toxin, epithelial cell function, and specifically monolayer formation, is intact in patients with HFS. Nevertheless, loss of ANTXR2-mediated signaling leads to collagen VI accumulation in the duodenum and abnormal extracellular matrix composition, which likely plays a role in development of PLE.


Subject(s)
Collagen/metabolism , Duodenum/metabolism , Hyaline Fibromatosis Syndrome/metabolism , Protein-Losing Enteropathies/metabolism , Receptors, Peptide/genetics , Antigens, Bacterial/chemistry , Bacterial Toxins/chemistry , CRISPR-Cas Systems , Consanguinity , Diarrhea/congenital , Extracellular Matrix/metabolism , Humans , Hyaline Fibromatosis Syndrome/genetics , Infant , Male , Microscopy, Electron , Mutation , Phenotype , Protein-Losing Enteropathies/genetics , Receptors, Peptide/deficiency , Signal Transduction
2.
Brain ; 140(3): 568-581, 2017 03 01.
Article in English | MEDLINE | ID: mdl-28364549

ABSTRACT

Cellular distribution and dynamics of mitochondria are regulated by several motor proteins and a microtubule network. In neurons, mitochondrial trafficking is crucial because of high energy needs and calcium ion buffering along axons to synapses during neurotransmission. The trafficking kinesin proteins (TRAKs) are well characterized for their role in lysosomal and mitochondrial trafficking in cells, especially neurons. Using whole exome sequencing, we identified homozygous truncating variants in TRAK1 (NM_001042646:c.287-2A > C), in six lethal encephalopathic patients from three unrelated families. The pathogenic variant results in aberrant splicing and significantly reduced gene expression at the RNA and protein levels. In comparison with normal cells, TRAK1-deficient fibroblasts showed irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration. This study confirms the role of TRAK1 in mitochondrial dynamics and constitutes the first report of this gene in association with a severe neurodevelopmental disorder.


Subject(s)
Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Brain Diseases/genetics , Brain Diseases/pathology , Mitochondria/metabolism , Mitochondrial Dynamics/genetics , Brain Diseases/diagnostic imaging , Brain Diseases/mortality , Cells, Cultured , Child, Preschool , Consanguinity , Family Health , Female , Fibroblasts/pathology , Fibroblasts/ultrastructure , Genetic Association Studies , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Infant , Magnetic Resonance Imaging , Male , Oxygen Consumption/genetics , Protein Transport/genetics , Transfection
3.
J Ultrasound Med ; 36(1): 149-154, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27933652

ABSTRACT

OBJECTIVES: The purpose of this study was to describe the characteristics and outcomes of fetuses with a diagnosis of nonobstructive diffuse dilated bowel loops. METHODS: We conducted a retrospective study of all pregnancies with fetal diagnosis of nonobstructive diffuse dilated bowel loops over 14 years in a large tertiary referral center. Fetomaternal and neonatal characteristics and outcomes were assessed. RESULTS: Seven fetuses had sonograms showing diffuse dilated bowel loops; none of them had intestinal obstruction after labor. The median gestational age at diagnosis was 33 weeks 1 day (range, 27 weeks-34 weeks 1 day). The median gestational age at delivery was 34 weeks 1 day (range, 32 weeks 4 days-39 weeks 1 day). Four cases had premature rupture of membranes beyond 32 weeks. Four among the 7 had gastrointestinal manifestations. Three cases presented with hematochezia, which resolved with conservative treatment. One fetus had intractable diarrhea, had a diagnosis of rare microvillus inclusion disease, and died of sepsis after 92 days. Not a single case of Hirschsprung disease was observed in our group. CONCLUSIONS: Nonobstructive diffuse dilated bowel loops diagnosed in the second half of pregnancy are associated with premature rupture of membranes and premature labor. As neonatal gastrointestinal complications may be anticipated, prenatal parental counseling with a neonatologist and pediatric gastroenterologist should be conducted.


Subject(s)
Gastrointestinal Diseases/diagnostic imaging , Gastrointestinal Tract/abnormalities , Ultrasonography, Prenatal , Adult , Female , Gastrointestinal Tract/diagnostic imaging , Gastrointestinal Tract/embryology , Humans , Infant, Newborn , Intestines/diagnostic imaging , Male , Pregnancy , Retrospective Studies
4.
Hum Mutat ; 37(8): 727-31, 2016 08.
Article in English | MEDLINE | ID: mdl-27060491

ABSTRACT

We investigated the cause of situs inversus totalis (SIT) in two siblings from a consanguineous family. Genotyping and whole-exome analysis revealed a homozygous change in NME7, resulting in deletion of an exon causing an in-frame deletion of 34 amino acids located in the second NDK domain of the protein and segregated with the defective lateralization in the family. NME7 is an important developmental gene, and NME7 protein is a component of the γ-tubulin ring complex. This mutation is predicted to affect the interaction of NME7 protein with this complex as it deletes the amino acids crucial for the binding. SIT associated with homozygous deletion in our patients is in line with Nme7(-/-) mutant mice phenotypes consisting of congenital hydrocephalus and SIT, indicating a novel human laterality patterning role for NME7. Further cases are required to elaborate the full human phenotype associated with NME7 mutations.


Subject(s)
Nucleoside-Diphosphate Kinase/genetics , Sequence Deletion , Situs Inversus/genetics , Amino Acid Sequence , Female , Humans , Male , Microtubule-Associated Proteins/metabolism , Models, Molecular , Nucleoside-Diphosphate Kinase/chemistry , Nucleoside-Diphosphate Kinase/metabolism , Pedigree , Protein Domains
5.
N Engl J Med ; 369(1): 54-65, 2013 Jul 04.
Article in English | MEDLINE | ID: mdl-23738510

ABSTRACT

BACKGROUND: Neutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity. METHODS: We clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase-chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene. RESULTS: All seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the child's ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of ß1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis. CONCLUSIONS: Defective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.).


Subject(s)
Immunologic Deficiency Syndromes/genetics , Neutropenia/congenital , Vesicular Transport Proteins/genetics , Animals , Child , Endosomes/metabolism , Homozygote , Humans , Immunologic Deficiency Syndromes/congenital , Immunologic Deficiency Syndromes/immunology , Mutation , Neutropenia/genetics , Neutrophils/physiology , Phenotype , Protein Transport , Vesicular Transport Proteins/metabolism , Zebrafish
6.
J Clin Immunol ; 33(8): 1395-402, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24142230

ABSTRACT

PURPOSE: To elucidate the relative role of the immune system and intestinal epithelium in the ethiopatogenesis of Celiac disease (CD). METHODS: A patient with childhood CD who underwent allogeneic bone marrow transplantation (BMT) for chronic myelogenous leukemia was followed for 5 years after resumption of gluten containing diet. Immunological memory to gliadin epitopes was assessed in the index patient and in 5 newly diagnosed CD patients by standard serology testing and by CFSE-based proliferation assays of peripheral blood CD4+ cells and of intestinal LPL towards gliadin-TTG antigens. Intestinal lymphocytes' origin was determined by combined immuno-histochemistry and fluorescent in-situ hybridiazation (FISH). RESULTS: Over 5 years of follow-up after receiving BMT from a HLA-matched woman and cessation of gluten-free diet, the patient has remained well, with negative periodic antibodies assays and unremarkable serial duodenal biopsies. In vitro proliferation assays showed lack of a memory response of the patient's peripheral blood and lamina propria CD4+ T-cells towards TTG, gliadin or TTG-treated gliadin, whereas memory responses were evident in the newly diagnosed CD patients. Immuno-FISH of post-BMT duodenal mucosa showed that the chromosomal phenotype of all the epithelial cells was XY. In contrast, CD45+ lymphocytic lineage cells were all donor-derived XX cells, presumably originating in the transplanted bone marrow and re-populating the intestinal wall. CONCLUSIONS: CD resolution following allogeneic BMT is associated with absent gliadin-specific memory response, and with a dichotomous lymphocyte-epithelial chimeric intestine. These observations suggest that the pathogenesis of CD is critically dependent upon the immune system rather than the epithelial compartment.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Celiac Disease/immunology , Celiac Disease/therapy , Gliadin/immunology , Immunologic Memory/immunology , Tissue Donors , Adult , Allografts , CD4-Positive T-Lymphocytes/transplantation , Celiac Disease/pathology , Cell Separation , Female , Follow-Up Studies , Humans , Intestinal Mucosa/cytology , Intestinal Mucosa/immunology , Male , Transplantation, Homologous , Treatment Outcome
7.
Prenat Diagn ; 32(1): 70-4, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22367672

ABSTRACT

OBJECTIVE: To increase awareness to the possibility of nemaline myopathy (NM) when abnormal prenatal ultrasound findings appear together with a carrier state for the common exon 55 deletion in the nebulin gene (NEB) of an Ashkenazi Jewish parent. METHODS: We describe four unrelated pregnancies with abnormal prenatal ultrasound findings resulting in the birth of newborns with NM, where one or both parents were of Ashkenazi Jewish origin. Data was collected retrospectively from the patients' medical files. Molecular analysis of NEB was performed on the DNA from the patients and parents. RESULTS: Prenatal ultrasound findings included polyhydramnios, decreased fetal movements, club feet, and arthrogryposis. A biopsy from two of the newborns was consistent with NM. In all of the newborns, the common NEB exon 55 deletion was detected in the heterozygote state and in three of them, a second novel mutation was found. CONCLUSIONS: Ultrasonographic findings suggestive of a myopathy and a carrier state for the NEB exon 55 deletion in one of the parents should trigger a thorough investigation for NM. The extreme size of NEB imposes great difficulties when searching for a second mutation, especially under the time constraints of an ongoing pregnancy.


Subject(s)
Gene Deletion , Genetic Carrier Screening/methods , Heterozygote , Muscle Proteins/genetics , Myopathies, Nemaline/diagnostic imaging , Myopathies, Nemaline/genetics , Ultrasonography, Prenatal , Adult , Codon, Nonsense , Exons/genetics , Female , Genetic Predisposition to Disease , Humans , Infant, Newborn , Jews/genetics , Male , Pedigree , Pregnancy
8.
Carcinogenesis ; 32(12): 1749-57, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21900211

ABSTRACT

Compelling evidences have rendered the tumor microenvironment a crucial determinant in cancer outcome. Activating transcription factor 3 (ATF3), a stress response transcription factor, is known to have a dichotomous role in tumor cells, acting either as a tumor suppressor or an oncogene in a context-dependent manner. However, its expression and possible role in the tumor microenvironment are hitherto unknown. Here we show that ATF3 is upregulated in the stromal compartment of several types of cancer. Accordingly, Cancer-associated fibroblasts (CAFs) ectopically expressing ATF3 proliferated faster as indicated by increased colony-forming capacity and promoted the growth of adjacent tumor cells when co-injected into nude mice. Utilizing a genome-wide profiling approach, we unraveled a robust gene expression program induced by ATF3 in CAFs. Focusing on a specific subset of genes, we found that the ability of stromal ATF3 to promote cancer progression is mediated by transcriptional repression of CLDN1 and induction of CXCL12 and RGS4. In addition, regulation of LIF, CLDN1, SERPINE2, HSD17B2, ITGA7 and PODXL by ATF3 mediated the increased proliferation capacity of CAFs. In sum, our findings implicate ATF3 as a novel stromal tumor promoter and suggest that targeting ATF3 pathway might be beneficial for anticancer therapy.


Subject(s)
Activating Transcription Factor 3/physiology , Neoplasms/genetics , Transcription, Genetic/physiology , Blotting, Western , Cell Compartmentation , Cell Line, Tumor , Cell Proliferation , Disease Progression , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/metabolism , Neoplasms/pathology , Real-Time Polymerase Chain Reaction , Stromal Cells/metabolism
9.
Isr Med Assoc J ; 12(6): 353-6, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20928989

ABSTRACT

BACKGROUND: HER2 is an important prognostic and predictive marker in invasive breast cancer. It is currently assessed by immunohistochemistry for protein over-expression and by fluorescence in situ hybridization for gene amplification. The immunohistochemistry-equivocal cases (2+) are currently retested by FISH to determine eligibility for trastuzumab treatment. Retesting by FISH significantly raises the cost of patient management and sometimes delays treatment. The 4B5 is a new, FDA-approved, rabbit monoclonal antibody for HER2 testing. OBJECTIVES: To examine the reliability of 4B5 IHC HER2 testing in cases found by CB11 IHC to be HER2 status equivocal. METHODS: Twenty-eight invasive breast cancer cases, with an equivocal HER2 status by CB11 IHC, were retested by the 4B5 antibody as well as by FISH analysis. The scoring was performed using the same guidelines as HercepTest and was correlated with the FISH ratio. RESULTS: Of the original 28 CB11 clone designated equivocal cases, 14 (50%) showed negative HER2 staining using the 4B5 clone (HercepTest score 0 and 1+). Five cases (18%) proved to be positive (HercepTest score 3+) and 9 cases (32%) remained equivocal (HercepTest score 2+). The corresponding FISH ratio results showed that all 4B5 negative cases were negative by FISH testing, with a negative predictive value of 100%; 4 of 5 of the 4B5-positive cases were positive by FISH testing, with a positive predictive value of 80%. One 4B5-positive case was borderline-high (2.2 ratio) by FISH. The correlation between 4B5 IHC and FISH was statistically significant (P = 0.0013) by chi-square test. CONCLUSIONS: Sequential testing by 4B5 IHC could greatly reduce the need for FISH testing in cases considered HER2 equivocal by CB11 IHC.


Subject(s)
Antibodies, Monoclonal , Breast Neoplasms/diagnosis , Immunohistochemistry/methods , In Situ Hybridization, Fluorescence , Receptor, ErbB-2/analysis , Biomarkers, Tumor/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Female , Gene Expression , Humans , Immunohistochemistry/standards , In Situ Hybridization, Fluorescence/standards , Pilot Projects , Receptor, ErbB-2/immunology , Receptor, ErbB-2/metabolism , Reproducibility of Results , Sensitivity and Specificity
10.
J Vasc Res ; 46(4): 299-310, 2009.
Article in English | MEDLINE | ID: mdl-19077391

ABSTRACT

OBJECTIVE: While both play a role in the transcriptional response of hypoxic endothelial cells (ECs), hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha differ in their transactivation sites, pointing at potentially different target genes. We studied the discrete and common effects of HIF-1alpha and HIF-2alpha on the cytokine expression and vasculogenic properties of ECs. METHODS AND RESULTS: H5V and bovine aortic ECs were transfected to express HIF-1alpha, HIF-2alpha or both. Overexpression of HIF-1alpha or HIF-2alpha and, to a greater extent, cotransfection of HIF-1alpha and HIF-2alpha resulted in EC activation, as revealed by analysis of the adhesion capacities and adhesion molecule surface expression of ECs. From the paracrine aspect, conditioned medium from HIF-expressing ECs was found to promote the migration and tube formation capacity of wild-type ECs, mostly following HIF-1alpha and HIF-2alpha coexpression. Antibody arrays revealed altered expression of multiple cytokines, pointing at consistent additive effects of HIF-1alpha and HIF-2alpha on angiogenic protein expression. Finally, HIF-1alpha and HIF-2alpha additively promoted vessel formation in vivo, as demonstrated by a Matrigel angiogenesis assay. CONCLUSION: Our results further clarify the functional roles of HIF-1alpha and HIF-2alpha in ECs and for the first time demonstrate a common contribution of HIF-1alpha and HIF-2alpha to vasculogenesis.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Endothelial Cells/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neovascularization, Physiologic , Angiogenic Proteins/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cattle , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cell Hypoxia , Cell Movement , Cells, Cultured , Culture Media, Conditioned/metabolism , Cytokines/metabolism , Endothelial Cells/immunology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Mice , Mice, Inbred C57BL , Paracrine Communication , Transfection
11.
Stem Cells ; 26(10): 2634-43, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18687993

ABSTRACT

Bone marrow stromal cells (BMSCs) contain progenitors capable of participating in postnatal angiogenesis. Hypoxia-inducible factors (HIFs) mediate endothelial activation by driving the expression of multiple angiogenic factors. We explored the potential of HIF-1alpha and HIF-2alpha modification in BMSCs, as a tool to improve cell-based angiogenic therapy. BMSCs were retrovirally transduced to express stable forms of HIF-1alpha and HIF-2alpha. HIF-1alpha and, to a greater extent, HIF-2alpha overexpression promoted differentiation of BMSCs to the endothelial lineage, evident by CD31 and Tie-2 expression and improved adhesive properties. Whereas chemotaxis toward stromal-derived factor 1 was higher in both HIF-alpha-expressing BMSCs, enhanced migration toward vascular endothelial growth factor was found only following overexpression of HIF-2alpha, supported by a robust expression of its receptor, Flk-1. HIF-alpha expression was associated with upregulation of angiogenic proteins and improved tube formation. Cytokine arrays of endothelial cells stimulated by medium collected from HIF-alpha-expressing BMSCs revealed further angiogenic activation and improved adhesive capacity. Eventually, delivery of HIF-2alpha-transduced BMSCs induced a more robust angiogenic response, compared with sham-transduced or HIF-1alpha-transduced BMSCs in the corneal micropocket angiogenesis model. Our results support the use of HIF-alpha genes, particularly HIF-2alpha, to augment the efficacy of future cell-based therapy. Disclosure of potential conflicts of interest is found at the end of this article.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Bone Marrow Cells/cytology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neovascularization, Physiologic , Stromal Cells/metabolism , Animals , Bone Marrow Cells/enzymology , Cell Adhesion , Cell Differentiation , Cell Movement , Cells, Cultured , Chemokine CXCL12/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Humans , Male , Mice , Paracrine Communication , Rats , Rats, Wistar , Receptors, CXCR4/metabolism , Retroviridae , Stromal Cells/cytology , Stromal Cells/enzymology , Transduction, Genetic , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism
12.
Am J Med Genet A ; 146A(23): 3054-7, 2008 Dec 01.
Article in English | MEDLINE | ID: mdl-18973246

ABSTRACT

We describe a newborn infant with multiple congenital skull fractures and intracranial hemorrhage. He also had multiple skin folds suggesting a connective tissue abnormality. Electron microscopy of the skin biopsy showed collagen abnormalities with a "hieroglyphic appearance." The analysis of the synthesis of collagen in the cultured dermal fibroblasts demonstrated an accumulation of procollagen I. Molecular analysis found a nonsense mutation Q225X in ADAMTS2 gene, which encodes procollagen I N-terminal proteinase. All these findings confirmed the diagnosis of Ehlers-Danlos syndrome type VIIC (MIM 225410). Family studies suggested a founder effect in Ashkenazi Jews originating from Belarus. Prenatal diagnosis in the subsequent pregnancy reassured the parents that the fetus was an unaffected carrier.


Subject(s)
Ehlers-Danlos Syndrome/complications , Ehlers-Danlos Syndrome/diagnostic imaging , Skull Fractures/congenital , Skull Fractures/etiology , ADAM Proteins/genetics , ADAMTS Proteins , Chorionic Villi Sampling , Ehlers-Danlos Syndrome/genetics , Ehlers-Danlos Syndrome/ultrastructure , Female , Fibrillar Collagens/ultrastructure , Humans , Infant, Newborn , Intracranial Hemorrhages/congenital , Intracranial Hemorrhages/diagnostic imaging , Intracranial Hemorrhages/etiology , Male , Mutation , Pedigree , Pregnancy , Premature Birth , Skin/ultrastructure , Skull Fractures/diagnostic imaging , Tomography, X-Ray Computed
13.
Ultrastruct Pathol ; 32(5): 199-205, 2008.
Article in English | MEDLINE | ID: mdl-18958793

ABSTRACT

The 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinoma, in which the carcinogen is administered systemically in drinking water, is the most comparable animal model to the development of human oral carcinoma. This is the first study to report the ultrastructural changes in this model. The most significant changes were observed in the carcinoma cells at the invasion front and included unique modifications in the basal lamina, presence of micropinocytotic vesicles (plasmalemmal caveolae), and emergence of cytoplasmic microfilaments featuring a parallel arrangement. The microfilaments, in both appearance and organization, were consistent with contractile microfilaments. These observations may be the morphological reflection of the phenotypic modifications occurring within the carcinoma cells, approaching smooth muscle differentiation.


Subject(s)
Neoplasms, Experimental/ultrastructure , Tongue Neoplasms/ultrastructure , 4-Nitroquinoline-1-oxide , Actin Cytoskeleton/ultrastructure , Animals , Basement Membrane/ultrastructure , Carcinoma, Squamous Cell/chemically induced , Carcinoma, Squamous Cell/ultrastructure , Caveolae/ultrastructure , Epithelial Cells/ultrastructure , Neoplasm Invasiveness , Neoplasms, Experimental/chemically induced , Rats , Tongue Neoplasms/chemically induced
14.
Hum Pathol ; 38(3): 435-42, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17217996

ABSTRACT

Many studies have been performed on chromosomal aberrations of extranodal marginal zone lymphomas. However, only a few have been published so far on ocular adnexal marginal zone lymphomas. We studied 18 cases of orbital lymphoid cell infiltrates. Using fluorescence in situ hybridization (FISH), we studied some of the most common chromosomal aberrations found in extranodal marginal zone lymphomas as: trisomies 3, and rearrangements of the 18q21 MALTI gene to detect the translocations t(11;18)(q21;q21) and t(14;18)(q32;q21)MALT1. Our goals were as follows: (1) study those aberrations in our material and compare them with the literature, (2) check their prognostic significance, and (3) check whether studying those aberrations with FISH can be used as a diagnostic tool to differentiate reactive from neoplastic infiltrates, in addition to immunohistochemistry and polymerase chain reaction. We found a high frequency of trisomies 3 (68%) and 18 (56.6%), the highest published so far in orbital lymphomas. On the other hand, no rearrangement was seen in any of our cases. The histologic picture and the clinical course were the same when there was one or more aberrations. As for the diagnostic significance, the presence of a prior, concurrent, or subsequent lymphoma in almost all the positive for aberrations cases suggests that either the orbital infiltrates in these cases are lymphomas, or they have, at least, a malignant potential or a genetic instability. Therefore, the demonstration of these numerical aberrations by FISH may be an additional sensitive, reliable, and relatively simple tool to differentiate reactive from neoplastic orbital lymphoid cell infiltrates when the immunohistochemistry and polymerase chain reaction, performed in a busy and routine-based histopathology laboratory, are unsatisfactory.


Subject(s)
Lymphoma/genetics , Lymphoma/pathology , Orbital Neoplasms/genetics , Orbital Neoplasms/pathology , Adult , Aged , Aged, 80 and over , Caspases/genetics , Chromosomes, Human, Pair 18/genetics , Chromosomes, Human, Pair 3/genetics , Female , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Male , Middle Aged , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Neoplasm Proteins/genetics , Polymerase Chain Reaction , Trisomy/genetics , Trisomy/pathology
15.
Int J Cardiol ; 171(1): 24-30, 2014 Jan 15.
Article in English | MEDLINE | ID: mdl-24315344

ABSTRACT

BACKGROUND: Familial restrictive cardiomyopathy (RCM) caused by a single gene mutation is the least common of the inherited cardiomyopathies. Only a few RCM-causing mutations have been described. Most mutations causing RCM are located in sarcomere protein genes which also cause hypertrophic cardiomyopathy (HCM). Other genes associated with RCM include the desmin and familial amyloidosis genes. In the present study we describe familial RCM with severe heart failure triggered by a de novo mutation in TTN, encoding the huge muscle filament protein titin. METHODS AND RESULTS: Family members underwent physical examination, ECG and Doppler echocardiogram studies. The family comprised 6 affected individuals aged 12-35 years. Linkage to candidate loci was performed, followed by gene sequencing. Candidate loci/gene analysis excluded 18 candidate genes but showed segregation with a common haplotype surrounding the TTN locus. Sequence analysis identified a de novo mutation within exon 266 of the TTN gene, resulting in the replacement of tyrosine by cysteine. p.Y7621C affects a highly conserved region in the protein within a fibronectin-3 domain, belonging to the A/I junction region of titin. No other disease-causing mutation was identified in cardiomyopathy genes by whole exome sequencing. CONCLUSIONS: Our study shows, for the first time, that mutations in TTN can cause restrictive cardiomyopathy. The giant filament titin is considered to be a determinant of a resting tension of the sarcomere and this report provides genetic evidence of its crucial role in diastolic function.


Subject(s)
Cardiomyopathy, Restrictive/diagnosis , Cardiomyopathy, Restrictive/genetics , Connectin/genetics , Mutation/genetics , Adolescent , Adult , Amino Acid Sequence , Child , Connectin/chemistry , Female , Humans , Male , Molecular Sequence Data , Pedigree , Protein Structure, Secondary , Young Adult
16.
PLoS One ; 6(6): e20916, 2011.
Article in English | MEDLINE | ID: mdl-21687694

ABSTRACT

BACKGROUND: Psoriasis is a complex disease at the cellular, genomic and genetic levels. The role of microRNAs in skin development was shown in a keratinocyte-specific Dicer knockout mouse model. Considering that two main characteristics of psoriasis are keratinocytes hyperproliferation and abnormal skin differentiation, we hypothesized that aberrant microRNA expression contributes to the psoriatic phenotype. Here, we describe the differential expression of miRNAs in psoriatic involved and uninvolved skin as compared to normal skin, revealing an additional aspect of this complex disorder. METHODOLOGY/PRINCIPAL FINDINGS: Expression arrays were used to compare microRNA expression in normal skin versus psoriatic involved and uninvolved skin. Fourteen differentially expressed microRNAs were identified, including hsa-miR-99a, hsa-miR-150, hsa-miR-423 and hsa-miR-197. The expression of these microRNAs was reevaluated by qPCR. IGF-1R, which is involved in skin development and the pathogenesis of psoriasis, is a predicted target of hsa-miR-99a. In an in situ hybridization assay, we found that IGF-1R and miR-99a are reciprocally expressed in the epidermis. Using a reporter assay, we found that IGF-1R is targeted by hsa-miR-99a. Moreover, over expression of miR-99a in primary keratinocytes down-regulates the expression of the endogenous IGF-1R protein. Over expression of miR-99a also inhibits keratinocyte proliferation and increases Keratin 10 expression. These findings suggest that overexpression of hsa-miR-99a in keratinocytes drives them towards differentiation. In primary keratinocytes grown in high Ca(++), miR-99a expression increases over time. Finally, we found that IGF1 increases the expression of miR-99a. CONCLUSIONS/SIGNIFICANCE: We identified several microRNAs that are expressed differentially in normal and psoriatic skin. One of these miRNAs is miR-99a that regulates the expression of IGF-1R. Moreover, miR-99a seems to play a role in the differentiation of keratinocytes. We suggest that miR-99a is one of the regulators of the IGF-1R signaling pathway in keratinocytes. Activation of IGF1 signaling results in elevation of miR-99a which represses the expression of IGF-1R.


Subject(s)
Gene Expression Profiling , MicroRNAs/genetics , Psoriasis/genetics , Psoriasis/metabolism , Receptor, IGF Type 1/metabolism , Skin/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Cell Differentiation/genetics , Cell Proliferation , HEK293 Cells , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Keratinocytes/pathology , Middle Aged , Phenotype , Psoriasis/pathology , Skin/cytology , Skin/pathology , Young Adult
17.
J Transplant ; 2011: 252387, 2011.
Article in English | MEDLINE | ID: mdl-21876779

ABSTRACT

Activation of the pancreatic lineage in the liver has been suggested as a potential autologous cell replacement therapy for diabetic patients. Transcription factors-induced liver-to-pancreas reprogramming has been demonstrated in numerous species both in vivo and in vitro. However, human-derived liver cells capable of acquiring the alternate pancreatic repertoire have never been characterized. It is yet unknown whether hepatic-like stem cells or rather adult liver cells give rise to insulin-producing cells. Using an in vitro experimental system, we demonstrate that proliferating adherent human liver cells acquire mesenchymal-like characteristics and a considerable level of cellular plasticity. However, using a lineage-tracing approach, we demonstrate that insulin-producing cells are primarily generated in cells enriched for adult hepatic markers that coexpress both albumin and mesenchymal markers. Taken together, our data suggest that adult human hepatic tissue retains a substantial level of developmental plasticity, which could be exploited in regenerative medicine approaches.

18.
J Mol Diagn ; 12(5): 687-96, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20595629

ABSTRACT

Subtypes of renal tumors have different genetic backgrounds, prognoses, and responses to surgical and medical treatment, and their differential diagnosis is a frequent challenge for pathologists. New biomarkers can help improve the diagnosis and hence the management of renal cancer patients. We extracted RNA from 71 formalin-fixed paraffin-embedded (FFPE) renal tumor samples and measured expression of more than 900 microRNAs using custom microarrays. Clustering revealed similarity in microRNA expression between oncocytoma and chromophobe subtypes as well as between conventional (clear-cell) and papillary tumors. By basing a classification algorithm on this structure, we followed inherent biological correlations and could achieve accurate classification using few microRNAs markers. We defined a two-step decision-tree classifier that uses expression levels of six microRNAs: the first step uses expression levels of hsa-miR-210 and hsa-miR-221 to distinguish between the two pairs of subtypes; the second step uses either hsa-miR-200c with hsa-miR-139-5p to identify oncocytoma from chromophobe, or hsa-miR-31 with hsa-miR-126 to identify conventional from papillary tumors. The classifier was tested on an independent set of FFPE tumor samples from 54 additional patients, and identified correctly 93% of the cases. Validation on qRT-PCR platform demonstrated high correlation with microarray results and accurate classification. MicroRNA expression profiling is a very effective molecular bioassay for classification of renal tumors and can offer a quantitative standardized complement to current methods of tumor classification.


Subject(s)
Kidney Neoplasms/classification , MicroRNAs/genetics , Humans , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Reverse Transcriptase Polymerase Chain Reaction
19.
Int J Biochem Cell Biol ; 42(8): 1355-62, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20619223

ABSTRACT

Distinguishing hepatocellular carcinoma from metastatic tumors in the liver is of great practical importance, with significant therapeutic and prognostic implications. This differential diagnosis can be difficult because metastatic cancers in the liver, especially adenocarcinomas, may mimic the morphology and immunoexpression of hepatocellular carcinoma. Biomarkers that are specifically expressed in either hepatocellular carcinoma or metastatic adenocarcinoma can therefore be useful diagnostic tools. To find such biomarkers, we studied microRNA expression in 144 tumor samples using custom microarrays. Hsa-miR-141 and hsa-miR-200c, microRNAs that promote epithelial phenotypes, had significantly higher levels in non-hepatic epithelial tumors. In contrast, endothelial-associated hsa-miR-126 showed higher expression levels in hepatocellular carcinomas. Combinations of these microRNAs accurately identified primary hepatocellular carcinoma from metastatic adenocarcinoma in the liver. These findings were validated using quantitative real-time PCR to measure microRNA expression in additional samples. Thus, the tissue-specific expression patterns of microRNAs make them useful biomarkers for the diagnosis of liver malignancies.


Subject(s)
Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms/diagnosis , Liver Neoplasms/genetics , Liver/metabolism , MicroRNAs/genetics , Carcinoma, Hepatocellular/pathology , Diagnosis, Differential , Humans , Liver/pathology , Liver Neoplasms/pathology , MicroRNAs/metabolism , Neoplasm Metastasis , ROC Curve , Reverse Transcriptase Polymerase Chain Reaction
20.
Pathol Res Pract ; 206(8): 578-84, 2010 Aug 15.
Article in English | MEDLINE | ID: mdl-20418022

ABSTRACT

For surgical pathologists, distinguishing whether a pulmonary neoplasm is primary or metastatic can be challenging, and current biomarkers do not always aid lung tumor classification. The tissue-associated expression of microRNA likely explains the remarkable finding that many tumors can be classified based solely on their microRNA expression signature. Here we show that microRNAs can serve as biomarkers for lung tumor classification. Using microRNA microarray data generated from 76 formalin-fixed, paraffin-embedded (FFPE) samples of either primary lung cancer or metastatic tumors to the lung, we have identified a set of microRNAs expressed differentially between these two groups. This set includes hsa-miR-182, which was most strongly over-expressed in the lung primary tumors, and hsa-miR-126, which was over-expressed in the metastatic tumors. The differential expression of this set of microRNAs was confirmed using qRT-PCR on a set of 54 samples. In light of our data, microRNA expression should be considered as a potential clinical biomarker for surgical pathologists faced with discerning the tumor type of an inscrutable lung neoplasm.


Subject(s)
Biomarkers, Tumor/genetics , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Diagnosis, Differential , Humans , MicroRNAs , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction
SELECTION OF CITATIONS
SEARCH DETAIL