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1.
Mod Rheumatol Case Rep ; 5(2): 342-346, 2021 07.
Article in English | MEDLINE | ID: mdl-33784948

ABSTRACT

Granulomatosis with polyangiitis (GPA), formerly known as Wegener's granulomatosis, is a small- and medium-vessel autoimmune vasculitis. Rare presentations of GPA can manifest as ophthalmologic and endocrinological deficits with sellar enhancement on imaging. While GPA typically presents distinct in appearance from other sellar pathologies, such as pituitary adenoma, we report the case of a 41-year-old woman with GPA of the pituitary that was initially diagnosed as pituitary macroadenoma with apoplexy and treated with two surgical resections without improvement of clinical symptoms. Pathology analysis of the second resection specimen revealed an inflammatory process consistent with GPA. After the pathologic and clinical diagnosis of GPA was established, treatment with steroid and steroid-sparing immunosuppressants resulted in improvements both on imaging and symptomatically. We discuss important aspects of the diagnosis and treatment of this rare presentation of GPA.


Subject(s)
Granulomatosis with Polyangiitis , Adenoma/diagnosis , Adult , Diagnosis, Differential , Female , Granulomatosis with Polyangiitis/diagnosis , Humans , Pituitary Neoplasms/diagnosis , Stroke/diagnosis
2.
Mol Immunol ; 133: 128-134, 2021 05.
Article in English | MEDLINE | ID: mdl-33657462

ABSTRACT

Systemic lupus erythematosus (SLE, lupus) is a chronic autoimmune disease characterized by loss of peripheral tolerance to nuclear self-antigens. It is increasingly recognized that aberrant T cell metabolism is a critical mediator of SLE immunopathology. Hypoxia inducible factor 1⍺ (HIF-1α) is a key transcription factor that regulates T cell metabolism in response to immune stimuli. T cell activation induces HIF-1α expression and transcriptional activation of HIF-responsive genes. HypoxamiRs are a group of microRNAs sensitive to HIF-1α transcriptional regulation that function to fine-tune the HIF-driven transcriptional program. The 'master' hypoxamiR, miR-210 is transcriptionally regulated by HIF-1α and negatively regulates HIF-1α activity. Although a key role for HIF-1α in has been described in a number of autoimmune and inflammatory diseases and abnormal microRNA expression profiles correlate with poor clinical outcome in a number of rheumatologic diseases, the expression and function of HIF-1α and miR-210 in lupus remains largely uncharacterized. Here we report HIF-1α and miR-210 differential and lineage-specific expression in systemic lupus erythematosus. We show that HIF-1α mRNA and protein is overexpressed in human lupus CD4+ cells but not in CD8+ or CD19+ cells. RORγt, was upregulated in human lupus lymphocytes while FoxP3 expression remained unchanged. We show that miR-210 expression in lupus-prone mice correlates with disease activity and is robustly and selectively upregulated in CD4+ cells from both human lupus patients and lupus-prone mice. Our results suggest that abnormal HIF-1α and miR-210 expression contributes to SLE immune pathology and that HIF-1α/miR-210 may represent a novel and important regulatory axis in SLE.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Lupus Erythematosus, Systemic/genetics , MicroRNAs/genetics , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Forkhead Transcription Factors/metabolism , Gene Expression Regulation/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , MicroRNAs/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism
3.
Ann Intern Med ; 172(11): ITC81-ITC96, 2020 06 02.
Article in English | MEDLINE | ID: mdl-32479157

ABSTRACT

Systemic lupus erythematosus (lupus) is characterized by aberrant activity of the immune system, leading to variable clinical symptoms. Lupus is more prevalent in African American women and women in other ethnic minority groups. Diagnosing, treating, and identifying novel therapies for lupus is challenging because of its genetic and phenotypic heterogeneity. Lupus nephritis is the most common target-organ manifestation and requires individualized care to minimize toxicity. A multidisciplinary approach to caring for pregnant patients with lupus is essential to optimize outcomes.


Subject(s)
Lupus Erythematosus, Systemic/diagnosis , Diagnosis, Differential , Humans , Lupus Erythematosus, Systemic/pathology , Lupus Erythematosus, Systemic/therapy
5.
Clin Immunol ; 162: 100-6, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26631756

ABSTRACT

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression primarily at the post-transcriptional level. Emerging evidence supports a regulatory role for miRNAs in the immune response and autoimmunity. In this work, we investigated the implication of miR-21 in the experimentally inducible bm12→B6 cGVHD model of systemic lupus erythematosus (SLE). cGVHD host mice deficient in miR-21 show a 2-fold reduction in splenomegaly, significantly reduced autoantibody titers and down-regulated components of the CD40:CD40L and CD28:CD80/86 co-stimulation pathways. Furthermore, we demonstrate that miR-21-deficient hosts have reduced CD4(+) IL-17(+) cell populations and an expanded CD4(+) CD25(+) FoxP3(+) cell compartment. We propose that miR-21 has a pluripotent role, serving to link distinct lymphocyte signaling pathways and acting as a "rheostat" for signals that promote B and T cell activation in lupus. Collectively, our experiments demonstrate that miR-21 deficiency in cGVHD host mice is sufficient to protect from lupus-like autoimmunity.


Subject(s)
Autoimmunity/genetics , Graft vs Host Disease , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , MicroRNAs/genetics , Animals , Disease Models, Animal , Flow Cytometry , Graft vs Host Disease/complications , Humans , Mice
7.
ACS Chem Biol ; 7(2): 403-10, 2012 Feb 17.
Article in English | MEDLINE | ID: mdl-22026461

ABSTRACT

Argonaute proteins are the core components of the microRNP/RISC. The biogenesis and function of microRNAs and endo- and exo- siRNAs are regulated by Ago2, an Argonaute protein with RNA binding and nuclease activities. Currently, there are no in vitro assays suitable for large-scale screening of microRNP/RISC loading modulators. We describe a novel in vitro assay that is based on fluorescence polarization of TAMRA-labeled RNAs loaded to human Ago2. Using this assay, we identified potent small-molecule inhibitors of RISC loading, including aurintricarboxylic acid (IC(50) = 0.47 µM), suramin (IC(50) = 0.69 µM), and oxidopamine HCL (IC(50) = 1.61 µM). Small molecules identified by this biochemical screening assay also inhibited siRNA loading to endogenous Ago2 in cultured cells.


Subject(s)
Argonaute Proteins/metabolism , Drug Evaluation, Preclinical/methods , RNA-Induced Silencing Complex/antagonists & inhibitors , RNA/analysis , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Cell Line , DNA/metabolism , Fluorescent Dyes/analysis , Humans , RNA/metabolism , RNA, Small Interfering/antagonists & inhibitors , RNA, Small Interfering/metabolism , RNA-Induced Silencing Complex/metabolism , Rhodamines/analysis
8.
EMBO Mol Med ; 3(10): 605-15, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21882343

ABSTRACT

MicroRNAs (miRNAs) have been implicated in B cell lineage commitment, regulation of T cell differentiation, TCR signalling, regulation of IFN signalling, and numerous other immunological processes. However, their function in autoimmunity, and specifically in systemic lupus erythematosus (SLE), remains poorly understood. B6.Sle123 is a spontaneous genetic mouse model of SLE characterized by autoantibody production, lymphosplenomegaly, and glomerulonephritis. We identified several differentially regulated miRNAs in B and T lymphocytes of B6.Sle123 mice. We found that miR-21 expression in lupus B and T cells is up-regulated and that in vivo silencing of miR-21 using a tiny seed-targeting LNA reversed splenomegaly, one of the cardinal manifestations of autoimmunity in B6.Sle123 mice, and de-repressed PDCD4 expression in vivo and in vitro. In addition, treatment with anti-miR-21 altered CD4/CD8 T cell ratios and reduced Fas receptor-expressing lymphocyte populations. Our study shows that tiny LNAs can be used to efficiently antagonize endogenous miRNAs in peripheral lymphocytes in vivo and in primary lymphocytes cultured ex vivo and can alter the course of a spontaneous genetic disease in mice.


Subject(s)
Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Gene Silencing , Lupus Erythematosus, Systemic/genetics , MicroRNAs/genetics , Splenomegaly/genetics , Splenomegaly/immunology , Aging/drug effects , Aging/pathology , Animals , Apoptosis Regulatory Proteins/metabolism , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , CD4-CD8 Ratio , Gene Expression Profiling , Gene Expression Regulation/drug effects , Gene Silencing/drug effects , Lupus Erythematosus, Systemic/complications , Lupus Erythematosus, Systemic/immunology , Lymphocyte Subsets/drug effects , Lymphocyte Subsets/immunology , Lymphocytes/drug effects , Lymphocytes/metabolism , Mice , Mice, Inbred C57BL , MicroRNAs/metabolism , Oligonucleotides/pharmacology , RNA-Binding Proteins/metabolism , Splenomegaly/complications , Splenomegaly/pathology , Transcription, Genetic/drug effects , fas Receptor/metabolism
9.
BMC Mol Biol ; 12: 19, 2011 Apr 29.
Article in English | MEDLINE | ID: mdl-21529364

ABSTRACT

BACKGROUND: Argonaute, the core component of the RNA induced silencing complex (RISC), binds to mature miRNAs and regulates gene expression at transcriptional or post-transcriptional level. We recently reported that Argonaute 2 (Ago2) also assembles into complexes with miRNA precursors (pre-miRNAs). These Ago2:pre-miRNA complexes are catalytically active in vitro and constitute non-canonical RISCs. RESULTS: The use of pre-miRNAs as guides by Ago2 bypasses Dicer activity and complicates in vitro RISC reconstitution. In this work, we characterized Ago2:pre-miRNA complexes and identified RNAs that are targeted by miRNAs but not their corresponding pre-miRNAs. Using these target RNAs we were able to recapitulate in vitro pre-miRNA processing and canonical RISC loading, and define the minimal factors required for these processes. CONCLUSIONS: Our results indicate that Ago2 and Dicer are sufficient for processing and loading of miRNAs into RISC. Furthermore, our studies suggest that Ago2 binds primarily to the 5'- and alternatively, to the 3'-end of select pre-miRNAs.


Subject(s)
Eukaryotic Initiation Factor-2/metabolism , MicroRNAs/metabolism , RNA-Induced Silencing Complex/metabolism , Animals , Argonaute Proteins , Base Sequence , Cell Line , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/isolation & purification , Humans , Mice , Molecular Sequence Data , RNA/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Ribonuclease III/metabolism
10.
Brain Res ; 1338: 122-30, 2010 Jun 18.
Article in English | MEDLINE | ID: mdl-20380813

ABSTRACT

We review the pertinent literature on methods used in high-throughput experimental identification of microRNA (miRNA) "targets" with emphasis on neurochemical studies. miRNAs are short regulatory noncoding RNAs that play important roles in the mammalian brain. The functions of miRNAs are related to their binding of RNAs including mRNAs. Since mammalian miRNAs tend to bind to target mRNAs via imperfect complementarity, understanding exactly which target mRNAs are recognized by which specific miRNAs is a challenge. Based on early experimental evidence, a set of "binding rules" for miRNAs has been described. These have focused on the 5' "seed" region of miRNAs binding to the 3' untranslated region of targeted mRNAs. Bioinformaticians have applied these algorithms for theoretical miRNA target prediction. To date, the different computational methods are not in agreement with each other and do not explain all miRNA targets as defined using high-throughput experimental methods. We consider these latter techniques which identify putative miRNA targets directly. Each experimental approach involves specific assumptions and potential technical pitfalls. Some of these direct experimental methods for miRNA target identification have used co-immunoprecipitation (RIP-Chip and others) and transfection-based experimental design. Topics related to experimentally identified miRNA targets are discussed, with special emphasis on studies pertinent to the mammalian brain.


Subject(s)
High-Throughput Screening Assays/methods , MicroRNAs/metabolism , Animals , Brain/metabolism , Computers , Humans , Mammals
11.
Nucleic Acids Res ; 37(22): 7533-45, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19808937

ABSTRACT

Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells.


Subject(s)
Eukaryotic Initiation Factor-2/metabolism , MicroRNAs/metabolism , RNA Precursors/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Substitution , Animals , Argonaute Proteins , Cell Line , Eukaryotic Initiation Factor-2/analysis , Eukaryotic Initiation Factor-2/genetics , Humans , Kinetics , Mice , Mice, Knockout , RNA Processing, Post-Transcriptional , RNA-Binding Proteins/genetics , RNA-Induced Silencing Complex/metabolism , Recombinant Fusion Proteins/metabolism , Ribonuclease III/genetics , Ribonucleoproteins/analysis
12.
RNA ; 13(10): 1787-92, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17720879

ABSTRACT

Argonaute (Ago) proteins bind to microRNA (miRNAs) and short interfering RNAs (siRNAs) and form the core components of effector complexes that mediate miRNA and siRNA function. Currently, there is a paucity of reliable antibodies against mammalian Ago proteins, thus precluding studies of endogenous Ago proteins from tissues. Here we report the development of 2A8, a novel anti-Ago monoclonal antibody that recognizes human and mouse Ago proteins and efficiently immunoprecipitates miRNAs. We report the characterization of 2A8 and its use to clone miRNAs from human brain and from preparations of human polymorphonuclear leukocytes (neutrophils), which revealed a prevalent miRNA with unusual features.


Subject(s)
Eukaryotic Initiation Factor-2/genetics , MicroRNAs/metabolism , Animals , Antibodies, Monoclonal/metabolism , Base Sequence , Cell Line , Humans , RNA Interference , Recombinant Proteins/metabolism
13.
Cell ; 129(6): 1141-51, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17524464

ABSTRACT

microRNAs (miRNAs) bind to Argonaute (Ago) proteins and inhibit translation or promote degradation of mRNA targets. Human let-7 miRNA inhibits translation initiation of mRNA targets in an m(7)G cap-dependent manner and also appears to block protein production, but the molecular mechanism(s) involved is unknown and the role of Ago proteins in translational regulation remains elusive. Here we identify a motif (MC) within the Mid domain of Ago proteins, which bears significant similarity to the m(7)G cap-binding domain of eIF4E, an essential translation initiation factor. We identify conserved aromatic residues within the MC motif of human Ago2 that are required for binding to the m(7)G cap and for translational repression but do not affect the assembly of Ago2 with miRNA or its catalytic activity. We propose that Ago2 represses the initiation of mRNA translation by binding to the m(7)G cap of mRNA targets, thus likely precluding the recruitment of eIF4E.


Subject(s)
Dinucleoside Phosphates/genetics , Eukaryotic Initiation Factor-2/genetics , Protein Biosynthesis , RNA, Messenger/metabolism , Amino Acid Motifs , Amino Acid Sequence , Argonaute Proteins , Base Sequence , Eukaryotic Initiation Factor-4E/chemistry , HeLa Cells , Humans , MicroRNAs/metabolism , Models, Genetic , Molecular Sequence Data , Protein Structure, Tertiary , Sequence Homology, Amino Acid
15.
Genes Dev ; 18(10): 1165-78, 2004 May 15.
Article in English | MEDLINE | ID: mdl-15131085

ABSTRACT

A new paradigm of gene expression regulation has emerged recently with the discovery of microRNAs (miRNAs). Most, if not all, miRNAs are thought to control gene expression, mostly by base pairing with miRNA-recognition elements (MREs) found in their messenger RNA (mRNA) targets. Although a large number of human miRNAs have been reported, many of their mRNA targets remain unknown. Here we used a combined bioinformatics and experimental approach to identify important rules governing miRNA-MRE recognition that allow prediction of human miRNA targets. We describe a computational program, "DIANA-microT", that identifies mRNA targets for animal miRNAs and predicts mRNA targets, bearing single MREs, for human and mouse miRNAs.


Subject(s)
MicroRNAs/genetics , Models, Genetic , Animals , Base Sequence , Binding Sites , Computational Biology , Computer Simulation , Genes, Reporter , HeLa Cells , Humans , Mice , MicroRNAs/chemistry , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Messenger/chemistry , RNA, Messenger/genetics , Software
16.
Trends Biochem Sci ; 28(10): 534-40, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14559182

ABSTRACT

A new paradigm of RNA-directed gene expression regulation has emerged recently, profound in scope but arresting in the apparent simplicity of its core mechanism. Cells express numerous small ( approximately 22 nucleotide) RNAs that act as specificity determinants to direct destruction or translational repression of their mRNA targets. These small RNAs arise from processing of double-stranded RNA by the Dicer nuclease and incorporate with proteins that belong to the Argonaute family. Small RNAs might also target and silence homologous DNA sequences. The immense potential of small RNAs as controllers of gene networks is just beginning to unfold.


Subject(s)
MicroRNAs/chemistry , RNA/metabolism , Animals , Caenorhabditis elegans , Chromatin/chemistry , Gene Expression Regulation , Gene Silencing , Humans , Protein Biosynthesis , Protein Folding , Protein Structure, Tertiary , RNA, Messenger/metabolism
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