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1.
Geroscience ; 41(3): 255-266, 2019 06.
Article in English | MEDLINE | ID: mdl-31280482

ABSTRACT

In the last decade, we have seen increasing evidence of the importance of structural nuclear proteins such as lamins in nuclear architecture and compartmentalization of genome function and in the maintenance of mechanical stability and genome integrity. With over 400 mutations identified in the LMNA gene (encoding for A-type lamins) associated with more than ten distinct degenerative disorders, the role of lamins as genome caretakers and the contribution of lamins dysfunction to disease are unarguable. However, the molecular mechanisms whereby lamins mutations cause pathologies remain less understood. Here, we review pathways and mechanisms recently identified as playing a role in the pathophysiology of laminopathies, with special emphasis in Hutchinson Gilford Progeria Syndrome (HGPS). This devastating incurable accelerated aging disease is caused by a silent mutation in the LMNA gene that generates a truncated lamin A protein "progerin" that exerts profound cellular toxicity and organismal decline. Patients usually die in their teens due to cardiovascular complications such as myocardial infarction or stroke. To date, there are no efficient therapies that ameliorate disease progression, stressing the need to understand molecularly disease mechanisms that can be targeted therapeutically. We will summarize data supporting that replication stress is a major cause of genomic instability in laminopathies, which contributes to the activation of innate immune responses to self-DNA that in turn accelerate the aging process.


Subject(s)
DNA/genetics , DNA/immunology , Genomic Instability/genetics , Immunity, Innate/genetics , Progeria/genetics , Progeria/immunology , DNA Damage/genetics , DNA Damage/immunology , DNA Mutational Analysis , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Humans , Immunity, Innate/immunology , Lamin Type A/genetics , Lamin Type A/immunology , Lamins/genetics , Lamins/immunology
2.
Article in English | MEDLINE | ID: mdl-30303754

ABSTRACT

The illegal use of glucocorticoids (GCs) as growth-promoters (GPs) is prohibited in farm animals in the European Union because the strong pharmacological activity of most synthetic GCs produces residues that are dangerous for human consumption. Among the alternative methods proposed to increase the efficacy of official controls, histology was the technique of choice in Italy on account of its high performance level. The aim of this study was to evaluate the reliability of immunohistochemistry (IHC) using anti-cleaved-Lamin A antibody to enhance the performance of the histological test applied to GC-related microscopic changes in the thymus. Veal calves (VC) and beef cattle (BC) were raised and both underwent different growth-promoting protocols or were left untreated. The morphology of the thymus parenchyma was evaluated for cortical atrophy with concurrent adipose tissue infiltration, and a score of 1 to 3 was attributed. A semi-quantitative IHC analysis was also performed by counting the number of positive thymocytes in 5 randomly selected high-power fields (HPFs). The distribution of the thymus atrophy scores was significantly different among the subgroups in both BC and VC. The IHC values were higher in untreated than in treated animals, for both BC and VC. The association between thymus atrophy score and IHC positivity showed higher median values in control than in treated animals (independently of the treatment protocol), for both BC and VC. Our data shows that IHC against anti-cleaved-Lamin A antibody is a reliable marker to detect illegal GC treatments, administered either alone or in association with other growth promoters, in both BC and VC. Combining IHC with the thymus atrophy score improves the accuracy of the histological method in correctly identifying treated animals and could represent a valuable, reproducible method to be applied to large-scale screening programmes.


Subject(s)
Food Analysis , Glucocorticoids/chemistry , Lamin Type A/analysis , Animals , Antibodies/immunology , Antigen-Antibody Reactions , Cattle , Glucocorticoids/pharmacology , Immunohistochemistry , Lamin Type A/immunology
3.
Oncotarget ; 7(49): 80252-80261, 2016 Dec 06.
Article in English | MEDLINE | ID: mdl-27835913

ABSTRACT

Lamin A/C proteins are major components of nuclear laminae and were encoded by the LMNA gene. Recent studies have found that in addition to provides nuclear-membrane strength; it also regulates the gene expression. Lamin A/C has been confirmed as an autoantigen in RA, SLE and vasculitis. Anti-Lamin A/C antibodies also have been found by indirect immunofluorescence method. In this study, we used various research methods to confirm Lamin A/C is an autoantigen in Han Chinese patients with confirmed Sjögren's syndrome (SS). To further investigate the relationship between the autoimmune disease antigens, we compared the amino acid sequence of Lamin A/C epitope and several common antigens' antigenic determinant. As a result, we found that Lamin A/C has similar epitopes with U1RNP. It means that the potential relationship exist between Lamin A/C and U1RNP. Clinical data we collected also showed that anti-Lamin A/C and anti-U1RNP antibodies always appear in same serum sample. Therefore, we speculated that cross-reaction may take place between antigen and potential antigen, which have similar epitope. Then, by epitope spreading, the potential antigen can be a new autoantigen. Our study provided a new thinking for further research about the relationship between autoantigens and their development mechanism in autoimmune diseases.


Subject(s)
Autoantibodies/blood , Lamin Type A/immunology , Sjogren's Syndrome/blood , Adult , Aged , Biomarkers/blood , Case-Control Studies , China , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Epitopes , Female , Humans , Male , Middle Aged , Ribonucleoprotein, U1 Small Nuclear/immunology , Sjogren's Syndrome/diagnosis , Sjogren's Syndrome/immunology , Young Adult
4.
Clin Exp Rheumatol ; 34(3 Suppl 97): S60-9, 2016.
Article in English | MEDLINE | ID: mdl-27214601

ABSTRACT

OBJECTIVES: Autoantibodies may play a role in the pathogenesis of primary vasculitides (PVs) like giant cell arteritis (GCA). We recently identified 3 cytoskeletal proteins (lamin C [laC], nuclear autoantigen of 14kD [NA14] and cytokeratin 15 [CK15]) as autoantigens in GCA and postulated a frequent autoantibody response against these antigens in PVs. METHODS: To test this hypothesis we studied a cohort of patients with PVs (n=61) and healthy individuals (n=27) for the presence of these autoantibodies using a recombinant cDNA expression library. To define their specifity for PV, we also examined patients with other autoimmune diseases such as rheumatoid arthritis (RA) and connective tissue diseases (CTD). RESULTS: We found no statistically significant differences in autoantibody responses between patients with PV and healthy controls, although there was a trend for an association between PVs and the occurrence of antibodies against laC and CK15. However, in patients with RA (n=33) or Sjögren's syndrome (SS, n=11) with vasculitides we observed more frequently autoantibodies against NA14, laC and CK15 compared to healthy controls. In patients with systemic lupus erythematosus (SLE, n=23) autoantibodies against laC were more frequent. The presence of autoantibodies in RA, SS and SLE was associated with systemic secondary vasculitis (SSV). In RA, laC- and NA14-seropositive patients were in a more advanced disease stage than seronegative patients with more frequent extraarticular manifestations (p=0.004). In SLE laC-autoantibody-positive patients had a higher SLE activity index (p<0.001). CONCLUSIONS: Serum autoantibodies against laC and NA14 are frequent in patients with RA and CTD and are associated with extensive disease and SSV. The potential pathogenic and prognostic role of these antibodies should be further investigated.


Subject(s)
Autoantibodies/blood , Autoantigens/immunology , Autoimmune Diseases/immunology , Keratin-15/immunology , Lamin Type A/immunology , Nuclear Proteins/immunology , Vasculitis/immunology , Adult , Aged , Female , Humans , Immunoglobulin G/blood , Male , Middle Aged
5.
Nucleus ; 5(5): 396-401, 2014.
Article in English | MEDLINE | ID: mdl-25482193

ABSTRACT

Nuclear lamins A/C control several critical cellular functions, e.g., chromatin organization, gene transcription, DNA replication, DNA damage responses, cell cycle progression, cell differentiation, and cell polarization during migration. However, few studies have addressed the role of lamins A/C in the control of the functions of immune cells. Recently, we have demonstrated that lamins A/C are induced in T cells upon antigen recognition. Lamins A/C enhance T cell responses by coupling the plasma membrane to the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex and the actin cytoskeleton. Here, we discuss the possible physiological relevance and functional context of lamin A/C in T cell activation and propose a model in which lamins A/C are key modulators of immune cell functions.


Subject(s)
Immune System , Lamin Type A/genetics , Lymphocyte Activation/genetics , T-Lymphocytes/immunology , Cell Differentiation/genetics , Cell Nucleus/genetics , Cell Nucleus/immunology , Chromatin/genetics , Humans , Lamin Type A/immunology , Lymphocyte Activation/immunology , Nuclear Envelope/genetics , Nuclear Envelope/immunology
7.
Sci Signal ; 7(322): ra37, 2014 Apr 22.
Article in English | MEDLINE | ID: mdl-24757177

ABSTRACT

In many cell types, nuclear A-type lamins regulate multiple cellular functions, including higher-order genome organization, DNA replication and repair, gene transcription, and signal transduction; however, their role in specialized immune cells remains largely unexplored. We showed that the abundance of A-type lamins was almost negligible in resting naïve T lymphocytes, but was increased upon activation of the T cell receptor (TCR). The increase in lamin-A was an early event that accelerated formation of the immunological synapse between T cells and antigen-presenting cells. Polymerization of F-actin in T cells is a critical step for immunological synapse formation, and lamin-A interacted with the linker of nucleoskeleton and cytoskeleton (LINC) complex to promote F-actin polymerization. We also showed that lamin-A expression accelerated TCR clustering and led to enhanced downstream signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, as well as increased target gene expression. Pharmacological inhibition of the ERK pathway reduced lamin-A-dependent T cell activation. Moreover, mice lacking lamin-A in immune cells exhibited impaired T cell responses in vivo. These findings underscore the importance of A-type lamins for TCR activation and identify lamin-A as a previously unappreciated regulator of the immune response.


Subject(s)
Actin Cytoskeleton/immunology , Actins/immunology , Immunological Synapses/immunology , Lamin Type A/immunology , Lymphocyte Activation/physiology , T-Lymphocytes/immunology , Actin Cytoskeleton/genetics , Actins/genetics , Animals , Humans , Immunological Synapses/genetics , Jurkat Cells , Lamin Type A/genetics , MAP Kinase Signaling System/genetics , MAP Kinase Signaling System/immunology , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/immunology , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/cytology
8.
Theriogenology ; 78(7): 1633-8, 2012 Oct 15.
Article in English | MEDLINE | ID: mdl-22980092

ABSTRACT

A precise, accurate, nonambiguous and high-throughput method is required to assess nuclear maturation of mammalian oocytes. The objectives of this study were to compare the efficiency and ease of use of a simplified fluorescence imaging (anti-lamin A/C and 4',6-diamidino-2-phenylindole [DAPI]) technique to the existing technique (aceto-orcein staining) for the evaluation of nuclear maturation of bovine oocytes, and to determine the kinetics of bovine oocyte maturation using an anti-lamin A/C-DAPI technique. In Experiment 1, oocytes were matured in vitro and stained with aceto-orcein and anti-lamin A/C-DAPI staining techniques. The proportions of oocytes lost during procedures and those that could not be classified (because of ambiguous morphology) during evaluation were lower (P < 0.0001) in oocytes stained with anti-lamin A/C-DAPI (9% and 2%) than those stained with aceto-orcein (31% and 13%), respectively. Anti-lamin A/C-DAPI was a quick procedure which could be completed within 7 h after completion of the maturation (compared with > 24 h for the aceto-orcein method). Furthermore, > 200 oocytes could be stained in one batch with anti-lamin A/C-DAPI technique. In Experiment 2, nuclear maturation kinetics of bovine oocytes at various time intervals (0, 6, 12, and 22 h) during in vitro maturation (IVM) was evaluated using the anti-lamin A/C-DAPI technique. Germinal vesicle, germinal vesicle breakdown, metaphase I, and metaphase II oocytes were predominant at 0, 6, 12, and 22 h of IVM, respectively. We concluded that the anti-lamin A/C-DAPI was an efficient and simple technique for nonambiguous evaluation of nuclear maturation status of large numbers of oocytes in a short interval.


Subject(s)
Cattle , Cell Nucleus/physiology , Fluorescent Dyes , Lamin Type A/immunology , Oocytes/ultrastructure , Oxazines , Animals , Cells, Cultured , Female , Fluorescent Antibody Technique/methods , Indoles , Metaphase , Oocytes/physiology , Staining and Labeling/methods
9.
Eur Respir J ; 39(6): 1405-14, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22005913

ABSTRACT

Anti-endothelial cell antibodies (AECAs) have been identified in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and in patients with idiopathic pulmonary arterial hypertension (iPAH). However, their target antigens remain poorly identified. Sera from 24 patients with SSc without PAH, 20 patients with SSc with PAH, 30 with iPAH and 12 healthy controls were collected. Target antigens were identified by two-dimensional electrophoresis and immunoblotting in protein extracts of human umbilical vein endothelial cells. Targeted antigens were identified by mass spectrometry. Serum immunoglobulin G from patients with SSc with or without PAH and patients with iPAH specifically recognised 110, 82 and 37 protein spots, respectively. Among others, target antigens of AECAs included lamin A/C, tubulin ß-chain and vinculin. One-dimension immunoblotting experiments confirmed the identification of lamin A/C and tubulin ß-chain. In conclusion, our results confirm the presence of AECA in patients with systemic sclerosis with and without pulmonary arterial hypertension and in those with idiopathic pulmonary arterial hypertension, and provide evidence for the identification of target antigens of these autoantibodies including lamin A/C and tubulin ß-chain.


Subject(s)
Autoantibodies/blood , Autoantibodies/immunology , Hypertension, Pulmonary/immunology , Scleroderma, Systemic/immunology , Adult , Autoantigens/immunology , Cells, Cultured , Female , Human Umbilical Vein Endothelial Cells/immunology , Humans , Hypertension, Pulmonary/blood , Immunoglobulin G/blood , Immunoglobulin G/immunology , Lamin Type A/immunology , Male , Microvessels/immunology , Middle Aged , Scleroderma, Systemic/blood , Tubulin/immunology , Vinculin/immunology
10.
J Immunol Methods ; 311(1-2): 139-52, 2006 Apr 20.
Article in English | MEDLINE | ID: mdl-16556448

ABSTRACT

Selective gene silencing by small interfering RNAs (siRNAs) has been shown to be an efficient method for the targeted manipulation of cellular functions. Chemical transfection reagents represent the current standard technique in siRNA duplex delivery into mammalian cells. However, when trying to manipulate cells isolated from patients in clinical approaches, chemical agents might cause unwanted side effects, such as allergic reactions, or interfere with other cellular functions. In this study we describe electroporation as a suitable and efficient method for the delivery of siRNA into monocyte-derived dendritic cells (moDCs). Using a fluorescein-labeled non-silencing siRNA duplex as a model system, we carefully investigated the effects of siRNA electroporation on moDCs' viability, phenotype, migratory capacity, and ability to induce T-cell mediated immune responses. Finally, by using a standard duplex directed against the nuclear Lamins A and C we were able to demonstrate an efficient knockdown of a cellular messenger RNA in electroporated moDCs. We therefore propose siRNA electroporation into moDCs as an efficient method to manipulate DC function at large cell numbers without the use of chemical transfection reagents. This new approach represents an advantage especially in the light of clinical trials.


Subject(s)
Dendritic Cells/physiology , Electroporation/methods , RNA Interference , RNA, Small Interfering/administration & dosage , Cell Movement , Dendritic Cells/immunology , Gene Silencing , Humans , Immunophenotyping , Lamin Type A/immunology , Lymphocyte Culture Test, Mixed , RNA/chemistry , RNA/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/immunology , Reverse Transcriptase Polymerase Chain Reaction
11.
Anal Biochem ; 344(2): 168-73, 2005 Sep 15.
Article in English | MEDLINE | ID: mdl-16095551

ABSTRACT

Polyethyleneimine (PEI) has been used previously as a nonviral DNA transfer vector. In this article, we demonstrate its use as a vehicle for transmembrane delivery of proteins in cell culture conditions. Linking proteins to PEI required no other treatment beyond mixing them with PEI. The bond between PEI and protein combined at optimal ratios was maintained in electrophoresis, even in the presence of 2.5% sodium dodecyl sulfate (SDS). The optimal time for delivery of proteins was determined to be 24 h. We have successfully delivered an Alexa 488-labeled avidin protein into human glioblastoma cells. A functional antibody against the nuclear protein lamin was delivered into human fibroblasts and reacted with lamin inside live cells. PEI-based delivery of antibodies and fluorescently labeled proteins can be used for fluorescent detection, tracking, and evaluation of cellular protein function in vivo.


Subject(s)
Cell Membrane/metabolism , Polyethyleneimine/chemistry , Protein Transport , Transfection/methods , Avidin/chemistry , Cells, Cultured , Electrophoresis, Agar Gel/methods , Fibroblasts , Fluorescent Dyes/chemistry , Glioblastoma , Humans , Hydrazines/chemistry , Infant, Newborn , Lamin Type A/immunology , Male , Tumor Cells, Cultured
12.
Biochim Biophys Acta ; 1671(1-3): 87-92, 2004 Mar 17.
Article in English | MEDLINE | ID: mdl-15026149

ABSTRACT

Using a phage-displayed peptide library, we have identified the epitope recognized by a new panel of five monoclonal antibodies (mAbs) raised against full-length recombinant human lamin A. The mAbs were found to recognize both lamin A and C by Western blotting and immunolocalization at the nuclear rim. A nine-amino acid consensus sequence PLLTYRFPP in the common immunoglobulin-like (Ig-like) domain of lamin A/C contains the binding site for all five mAbs. Three-dimensional structure of the Ig-like domain of lamin A/C shows this sequence is a complete beta-strand. This sequence includes arginine-482 (R482) which is mutated in most cases of Dunnigan-type familial partial lipodystrophy (FPLD). R482 may be part of an interaction site on the surface of lamin A/C for lamin-binding proteins associated with lipodystrophy.


Subject(s)
Antibodies, Monoclonal/immunology , Epitopes , Lamin Type A/genetics , Lamin Type A/immunology , Lipodystrophy/genetics , Protein Structure, Secondary , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Cells, Cultured , Epitope Mapping , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Lamin Type A/metabolism , Lipodystrophy/immunology , Models, Molecular , Molecular Sequence Data , Muscle, Skeletal/metabolism , Peptide Library , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Sequence Alignment
13.
J Cell Biochem ; 91(4): 730-9, 2004 Mar 01.
Article in English | MEDLINE | ID: mdl-14991764

ABSTRACT

The A-type lamins are localized in the interior of the nucleus as well as on the nuclear periphery. In this study, we have characterized a monoclonal antibody LA-2F9 produced against recombinant rat lamin A which stains a subpopulation of various cell types in a pattern of small nucleoplasmic foci that are unusually susceptible to mild detergent/salt extraction. The specific reactivity of mAb LA-2F9 towards lamins was confirmed by immunoblotting of HeLa and C3H10T(1/2) whole cell lysates and nuclear lysates. The epitope for LA-2F9 was narrowed down to amino acid residues 268-278 (SAKLDNARQSA). To check whether the appearance of lamin foci was cell-cycle-dependent, C3H10T(1/2) cells were serum-starved and then refed to trigger cells to enter the G(1) phase of the cell-cycle. The intensity of staining increased 3.5-fold within 6 h of refeeding, when the maximum number of cells were labeled with LA-2F9. We also checked whether the LA-2F9 foci colocalized with nuclear proteins known to be distributed in small foci such as hnRNPs, snRNPs, SC-35, and p80 coilin, but did not find evidence of colocalization. Our studies suggest that LA-2F9 has a novel and specific reactivity towards detergent-susceptible lower order lamin structures that are likely to be assembly intermediates.


Subject(s)
Antibodies, Monoclonal/immunology , Cell Nucleus/metabolism , Detergents/pharmacology , Lamin Type A/analysis , Lamin Type A/immunology , Animals , Antibodies, Monoclonal/isolation & purification , Antibody Specificity , Cell Nucleus/drug effects , Epitope Mapping , G1 Phase , Humans , Lamin Type A/chemistry , Mice , Microscopy, Fluorescence , Nuclear Proteins/analysis , Nuclear Proteins/chemistry , Nuclear Proteins/immunology , Rats
14.
Brain Res ; 976(1): 9-21, 2003 Jun 20.
Article in English | MEDLINE | ID: mdl-12763617

ABSTRACT

Monoclonal antibody J1-31 was raised against plaque materials taken from brains of patients who had suffered from multiple sclerosis (MS). Preliminary characterization of the antigen revealed it to be a protein of M(w) 68-70 kDa with both a cytoplasmic and nuclear localization. Here we report the results of isolation and peptide sequencing of the antigen from human brains, and immunocytochemical analysis of the antigen in F98 glioma cells. Purification and peptide sequencing indicate that the antibody recognizes a form of glial fibrillary acidic protein, possibly a phosphorylated variant. However, confocal immunocytochemistry and western analysis of F98 glioma cells raise the possibility that it also recognizes a phosphorylated epitope found in nuclear lamins. Analysis of the expression of the J1-31 epitope in F98 cells with respect to time in culture, cell density, and DNA synthesis showed a developmental relationship: cells that were engaged in rapid growth and DNA synthesis exhibited strong J1-31 staining in nuclei, whereas quiescent cells did not. We conclude that mAB J1-31 remains a useful antibody for studying multiple sclerosis, and is likely to prove useful in studies of the dynamics of nuclear lamins, particularly in models for wound-healing.


Subject(s)
Antibodies, Monoclonal/immunology , Glial Fibrillary Acidic Protein/metabolism , Lamins/metabolism , Amino Acid Sequence , Brain/metabolism , Brain/pathology , Brain/ultrastructure , Electrophoresis, Polyacrylamide Gel , Epitopes , Glial Fibrillary Acidic Protein/chemistry , Glial Fibrillary Acidic Protein/immunology , Humans , Immunohistochemistry , Lamin Type A/immunology , Lamin Type A/metabolism , Lamin Type B/immunology , Lamin Type B/metabolism , Lamins/immunology , Microscopy, Confocal , Molecular Sequence Data , Multiple Sclerosis/pathology , Peptide Fragments/chemistry , Tumor Cells, Cultured
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