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1.
Elife ; 92020 05 15.
Article in English | MEDLINE | ID: mdl-32410729

ABSTRACT

The identification of autoantigens remains a critical challenge for understanding and treating autoimmune diseases. Autoimmune polyendocrine syndrome type 1 (APS1), a rare monogenic form of autoimmunity, presents as widespread autoimmunity with T and B cell responses to multiple organs. Importantly, autoantibody discovery in APS1 can illuminate fundamental disease pathogenesis, and many of the antigens found in APS1 extend to more common autoimmune diseases. Here, we performed proteome-wide programmable phage-display (PhIP-Seq) on sera from a cohort of people with APS1 and discovered multiple common antibody targets. These novel APS1 autoantigens exhibit tissue-restricted expression, including expression in enteroendocrine cells, pineal gland, and dental enamel. Using detailed clinical phenotyping, we find novel associations between autoantibodies and organ-restricted autoimmunity, including a link between anti-KHDC3L autoantibodies and premature ovarian insufficiency, and between anti-RFX6 autoantibodies and diarrheal-type intestinal dysfunction. Our study highlights the utility of PhIP-Seq for extensively interrogating antigenic repertoires in human autoimmunity and the importance of antigen discovery for improved understanding of disease mechanisms.


The immune system uses antibodies to fight microbes that cause disease. White blood cells pump antibodies into the bloodstream, and these antibodies latch onto bacteria and viruses, targeting them for destruction. But sometimes, the immune system gets it wrong. In autoimmune diseases, white blood cells mistakenly make antibodies that target the body's own tissues. Detecting these 'autoantibodies' in the blood can help doctors to diagnose autoimmune diseases. But the identities and targets of many autoantibodies remain unknown. In one rare disease, called autoimmune polyendocrine syndrome type 1 (APS-1), a faulty gene makes the immune system much more likely to make autoantibodies. People with this disease can develop an autoimmune response against many different healthy organs. Although APS-1 is rare, some of the autoantibodies made by individuals with the disease are the same as the ones in more common autoimmune diseases, like type 1 diabetes. Therefore, investigating the other autoantibodies produced by individuals with APS-1 could reveal the autoantibodies driving other autoimmune diseases. Autoantibodies bind to specific regions of healthy proteins, and one way to identify them is to use hundreds of thousands of tiny viruses in a technique called proteome-wide programmable phage-display, or PhIP-Seq. Each phage carries one type of protein segment. When mixed with blood serum from a patient, the autoantibodies stick to the phages that carry the target proteins for that autoantibody. These complexes can be isolated using biochemical techniques. Sequencing the genes of these phages then reveals the identity of the autoantibodies' targets. Using this technique, Vazquez et al successfully pulled 23 known autoantibodies from the serum of patients with APS-1. Then, experiments to search for new targets began. This revealed many new autoantibodies, targeting proteins found only in specific tissues. They included one that targets a protein found on cells in the gut, and another that targets a protein found on egg cells in the ovaries. Matching the PhIP-Seq data to patient symptoms confirmed that these new antibodies correlate with the features of specific autoimmune diseases. For example, patients with antibodies that targeted the gut protein were more likely to have gut symptoms, while patients with antibodies that targeted the egg cell protein were more likely to have problems with their ovaries. Further investigations using PhIP-Seq could reveal the identities of even more autoantibodies. This might pave the way for new antibody tests to diagnose autoimmune diseases and identify tissues at risk of damage. This could be useful not only for people with APS-1, but also for more common autoimmune diseases that target the same organs.


Subject(s)
Autoantibodies/blood , Autoantigens/blood , Autoimmunity , Cell Surface Display Techniques , Polyendocrinopathies, Autoimmune/blood , Proteome , Proteomics , Acid Phosphatase/blood , Acid Phosphatase/immunology , Autoantigens/immunology , Biomarkers/blood , Female , HEK293 Cells , Humans , Male , Peptide Library , Polyendocrinopathies, Autoimmune/diagnosis , Polyendocrinopathies, Autoimmune/immunology , Proteins/immunology , Regulatory Factor X Transcription Factors/blood , Regulatory Factor X Transcription Factors/immunology
3.
Diabetologia ; 61(1): 168-181, 2018 01.
Article in English | MEDLINE | ID: mdl-28852792

ABSTRACT

AIMS/HYPOTHESIS: Identification of a pancreatic neuro-insular network in mice suggests that a similar integration of islets and nerves may be present in the human pancreas. To characterise the neuro-insular network and the intra-pancreatic ganglia in a clinically related setting, we examined human pancreases in health and with fatty infiltration via 3-dimensional (3D) histology and compared the human pancreatic microenvironment with its counterpart in mice. METHODS: Human pancreatic specimens from individuals with normal BMI, high BMI (≥ 25) and type 2 diabetes were used to investigate the neuro-insular network. Transparent specimens were prepared by tissue clearing for transmitted light and deep-tissue fluorescence imaging to simultaneously visualise infiltrated adipocytes, islets and neurovascular networks. RESULTS: High-definition images of human islets reveal that both the sympathetic and parasympathetic nerves enter the islet core and reside in the immediate microenvironment of islet cells. Around the islets, the neuro-insular network is visualised with 3D histology to identify the intra-pancreatic ganglia (peri-lobular and intra-parenchymal ganglia) and the islet-ganglionic association. In humans, but not in mice, pancreatic fatty infiltration (BMI dependent) features adipocytes infiltrating into the parenchyma and accumulating in the peri-lobular space, in which the peri-lobular ganglia also reside. We identified the formation of adipose-ganglionic complexes in the peri-lobular space and enlargement of ganglia around adipocytes. In the specimen from the individual with type 2 diabetes, an increase in the number of nerve projections from the intra-parenchymal ganglia is associated with severe fatty infiltration. CONCLUSIONS/INTERPRETATION: We present new perspectives of human pancreas and islet innervation via 3D histology. Our results strongly suggest that fatty infiltration in the human pancreas creates a neurotrophic microenvironment and promotes remodelling of pancreatic innervation.


Subject(s)
Pancreas/metabolism , Adipocytes/metabolism , Animals , Body Mass Index , Diabetes Mellitus, Type 2/metabolism , Humans , Islets of Langerhans/metabolism , Mice , Obesity/metabolism , Sympathetic Nervous System/metabolism
4.
Cell Metab ; 22(6): 997-1008, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26525534

ABSTRACT

Catecholamines promote lipolysis both in brown and white adipocytes, whereas the same stimuli preferentially activate thermogenesis in brown adipocytes. Molecular mechanisms for the adipose-selective activation of thermogenesis remain poorly understood. Here, we employed quantitative phosphoproteomics to map global and temporal phosphorylation profiles in brown, beige, and white adipocytes under ß3-adrenenoceptor activation and identified kinases responsible for the adipose-selective phosphorylation profiles. We found that casein kinase2 (CK2) activity is preferentially higher in white adipocytes than brown/beige adipocytes. Genetic or pharmacological blockade of CK2 in white adipocytes activates the thermogenic program in response to cAMP stimuli. Such activation is largely through reduced CK2-mediated phosphorylation of class I HDACs. Notably, inhibition of CK2 promotes beige adipocyte biogenesis and leads to an increase in whole-body energy expenditure and ameliorates diet-induced obesity and insulin resistance. These results indicate that CK2 is a plausible target to rewire the ß3-adrenenoceptor signaling cascade that promotes thermogenesis in adipocytes.


Subject(s)
Adipose Tissue, Brown/metabolism , Casein Kinase II/metabolism , Energy Metabolism , Phosphopeptides/analysis , Proteomics , Adipose Tissue, Brown/drug effects , Adipose Tissue, White/drug effects , Adipose Tissue, White/metabolism , Animals , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/genetics , Cyclic AMP/metabolism , Energy Metabolism/drug effects , Histone Deacetylases/chemistry , Histone Deacetylases/metabolism , Ion Channels/genetics , Ion Channels/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Naphthyridines/pharmacology , Norepinephrine/pharmacology , Obesity/etiology , Oxides/pharmacology , Phenazines , Receptors, Adrenergic, beta-3/metabolism , Signal Transduction , Thermogenesis/drug effects , Uncoupling Protein 1 , Vanadium Compounds/pharmacology
5.
Proc Natl Acad Sci U S A ; 112(9): 2888-93, 2015 Mar 03.
Article in English | MEDLINE | ID: mdl-25695968

ABSTRACT

Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic ß cells, and variants in genes encoding several Gi-GPCRs--including the α-2a adrenergic receptor, ADRA2A--increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total ß-cell mass, and the role of Gi-GPCRs in establishing ß-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates ß-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic ß cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal ß cells decreased ß-cell proliferation, reduced adult ß-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal ß-cell proliferation, increased adult ß-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult ß cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of ß-cell replication. These studies link Gi-GPCR signaling to ß-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase ß-cell mass in patients with diabetes.


Subject(s)
Cell Proliferation , Diabetes Mellitus, Type 2/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Insulin-Secreting Cells/metabolism , Receptors, Adrenergic, alpha-2/metabolism , Signal Transduction , Animals , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/pathology , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Glucose/genetics , Glucose/metabolism , Insulin-Secreting Cells/pathology , Mice , Mice, Transgenic , Receptors, Adrenergic, alpha-2/genetics
6.
J Clin Invest ; 124(9): 4093-101, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25133424

ABSTRACT

Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic ß cells for people with diabetes and for targeting menin-sensitive endocrine tumors.


Subject(s)
Islets of Langerhans/cytology , Proto-Oncogene Proteins/physiology , ras Proteins/physiology , Adult , Animals , Cell Proliferation , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Male , Mice , Middle Aged , Phosphorylation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Signal Transduction , Tumor Suppressor Proteins/physiology
7.
Nat Biotechnol ; 32(1): 76-83, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24240391

ABSTRACT

Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.


Subject(s)
Ciliary Neurotrophic Factor/administration & dosage , Diabetes Mellitus/drug therapy , Epidermal Growth Factor/administration & dosage , Insulin-Secreting Cells/drug effects , Acinar Cells/drug effects , Acinar Cells/pathology , Animals , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Ciliary Neurotrophic Factor/genetics , Diabetes Mellitus/genetics , Epidermal Growth Factor/genetics , Hyperglycemia/drug therapy , Insulin-Secreting Cells/pathology , Mice , Mice, Inbred NOD/genetics , Signal Transduction
8.
PLoS One ; 7(11): e49452, 2012.
Article in English | MEDLINE | ID: mdl-23166672

ABSTRACT

Brown adipose tissue (BAT) dissipates chemical energy and generates heat to protect animals from cold and obesity. Rodents possess two types of UCP-1 positive brown adipocytes arising from distinct developmental lineages: "classical" brown adipocytes develop during the prenatal stage whereas "beige" or "brite" cells that reside in white adipose tissue (WAT) develop during the postnatal stage in response to chronic cold or PPARγ agonists. Beige cells' inducible characteristics make them a promising therapeutic target for obesity treatment, however, the relevance of this cell type in humans remains unknown. In the present study, we determined the gene signatures that were unique to classical brown adipocytes and to beige cells induced by a specific PPARγ agonist rosiglitazone in mice. Subsequently we applied the transcriptional data to humans and examined the molecular signatures of human BAT isolated from multiple adipose depots. To our surprise, nearly all the human BAT abundantly expressed beige cell-selective genes, but the expression of classical brown fat-selective genes were nearly undetectable. Interestingly, expression of known brown fat-selective genes such as PRDM16 was strongly correlated with that of the newly identified beige cell-selective genes, but not with that of classical brown fat-selective genes. Furthermore, histological analyses showed that a new beige cell marker, CITED1, was selectively expressed in the UCP1-positive beige cells as well as in human BAT. These data indicate that human BAT may be primary composed of beige/brite cells.


Subject(s)
Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/metabolism , Ion Channels/metabolism , Mitochondrial Proteins/metabolism , Adipose Tissue, White/cytology , Animals , Apoptosis Regulatory Proteins , DNA-Binding Proteins/metabolism , Humans , Immunohistochemistry , Magnetic Resonance Imaging , Mice , Mice, Inbred C57BL , Microarray Analysis , Myogenic Regulatory Factor 5/metabolism , Nuclear Proteins/metabolism , Oligonucleotide Array Sequence Analysis , PPAR gamma/agonists , Rosiglitazone , Thiazolidinediones , Trans-Activators , Transcription Factors/metabolism , Uncoupling Protein 1
9.
Dis Model Mech ; 4(2): 268-76, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21135059

ABSTRACT

Transient expression of the transcription factor neurogenin-3 marks progenitor cells in the pancreas as they differentiate into islet cells. We developed a transgenic mouse line in which the surrogate markers secreted alkaline phosphatase (SeAP) and enhanced green florescent protein (EGFP) can be used to monitor neurogenin-3 expression, and thus islet cell genesis. In transgenic embryos, cells expressing EGFP lined the pancreatic ducts. SeAP was readily detectable in embryos, in the media of cultured embryonic pancreases and in the serum of adult animals. Treatment with the γ-secretase inhibitor DAPT, which blocks Notch signaling, enhanced SeAP secretion rates and increased the number of EGFP-expressing cells as assayed by fluorescence-activated cell sorting (FACS) and immunohistochemistry in cultured pancreases from embryos at embryonic day 11.5, but not in pancreases harvested 1 day later. By contrast, treatment with growth differentiation factor 11 (GDF11) reduced SeAP secretion rates. In adult mice, partial pancreatectomy decreased, whereas duct ligation increased, circulating SeAP levels. This model will be useful for studying signals involved in islet cell genesis in vivo and developing therapies that induce this process.


Subject(s)
Diabetes Mellitus/therapy , Islets of Langerhans/embryology , Models, Animal , Organogenesis , Alkaline Phosphatase/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Bone Morphogenetic Proteins/metabolism , Cell Lineage , Fetus/metabolism , Green Fluorescent Proteins/metabolism , Growth Differentiation Factors/metabolism , Humans , Islets of Langerhans/pathology , Mice , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Receptors, Notch/metabolism , Signal Transduction , Transgenes/genetics
10.
Nature ; 463(7282): 775-80, 2010 Feb 11.
Article in English | MEDLINE | ID: mdl-20148032

ABSTRACT

Insulin from the beta-cells of the pancreatic islets of Langerhans controls energy homeostasis in vertebrates, and its deficiency causes diabetes mellitus. During embryonic development, the transcription factor neurogenin 3 (Neurog3) initiates the differentiation of the beta-cells and other islet cell types from pancreatic endoderm, but the genetic program that subsequently completes this differentiation remains incompletely understood. Here we show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurog3. Mice lacking Rfx6 failed to generate any of the normal islet cell types except for pancreatic-polypeptide-producing cells. In human infants with a similar autosomal recessive syndrome of neonatal diabetes, genetic mapping and subsequent sequencing identified mutations in the human RFX6 gene. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors that coordinate pancreatic islet development in both mice and humans. Rfx6 could prove useful in efforts to generate beta-cells for patients with diabetes.


Subject(s)
Cell Differentiation , DNA-Binding Proteins/metabolism , Insulin/biosynthesis , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/deficiency , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA Mutational Analysis , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Diabetes Mellitus/congenital , Diabetes Mellitus/genetics , Diabetes Mellitus/metabolism , Diabetes Mellitus/pathology , Embryo, Mammalian/metabolism , Female , Fetus/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Genes, Recessive/genetics , Genetic Testing , Humans , Infant, Newborn , Islets of Langerhans/embryology , Male , Mice , NIH 3T3 Cells , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Organ Specificity , Regulatory Factor X Transcription Factors , Syndrome , Transcription Factors/deficiency , Transcription Factors/genetics
11.
Dev Biol ; 280(1): 111-21, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15766752

ABSTRACT

Embryonic Hedgehog signaling is essential for proper tissue morphogenesis and organ formation along the developing gastrointestinal tract. Hedgehog ligands are expressed throughout the endodermal epithelium at early embryonic stages but excluded from the region that will form the pancreas. Ectopic activation of Hedgehog signaling at the onset of pancreas development has been shown to inhibit organ morphogenesis. In contrast, Hedgehog signaling components are found within pancreatic tissue during subsequent stages of development as well as in the mature organ, indicating that a certain level of pathway activation is required for normal organ development and function. Here, we ectopically activate the Hedgehog pathway midway through pancreas development via expression of either Sonic (Shh) or Indian Hedgehog (Ihh) under control of the human Pax4-promoter. Similar pancreatic defects are observed in both Pax4-Shh and Pax4-Ihh transgenic lines, suggesting that regulation of the overall level of Hedgehog activity is critical for proper pancreas development. We also show that Hedgehog signaling controls mesenchymal vs. epithelial tissue differentiation and that pathway activation impairs formation of epithelial progenitors. Thus, tight control of Hedgehog pathway activity throughout embryonic development ensures proper pancreas organogenesis.


Subject(s)
Epithelial Cells/physiology , Morphogenesis/physiology , Pancreas/cytology , Signal Transduction , Trans-Activators/metabolism , Animals , Endocrine System/embryology , Epithelial Cells/cytology , Hedgehog Proteins , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , In Situ Hybridization , Mesoderm/cytology , Mesoderm/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Pancreas/abnormalities , Pancreas/embryology , Pancreas/metabolism , Promoter Regions, Genetic , Stem Cells/cytology , Stem Cells/metabolism , Trans-Activators/genetics , Transcription Factors/genetics , Transcription Factors/metabolism
12.
J Biol Chem ; 278(19): 17130-40, 2003 May 09.
Article in English | MEDLINE | ID: mdl-12604598

ABSTRACT

Homeodomain transcription factor Nkx2.2 is required for the final differentiation of the beta-cells in the pancreas and for the production of insulin. Nkx2.2 is expressed in islet cell precursors during pancreatic development and persists in a subset of mature islet cells including all beta-cells. To understand the mechanisms regulating the expression of Nkx2.2 in these different cell populations, we outlined the structure of the mouse nkx2.2 gene and identified regions that direct cell type-specific expression. The nkx2.2 gene has two noncoding alternative first exons (exons 1a and 1b). In transgenic mice, sequences upstream from exon 1a directed expression predominantly in mature islet cells. Within this exon 1a promoter, cooperative interactions between HNF3 and basic helix-loop-helix factors neurogenin-3 or NeuroD1 binding to adjacent sites played key roles in its islet cell-specific expression. In contrast, sequences upstream from exon 1b restricted expression specifically to islet cell precursors. These studies reveal distinct mechanisms for directing the expression of a key differentiation factor in precursors versus mature islet cells.


Subject(s)
Cell Differentiation/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Islets of Langerhans/physiology , Transcription Factors/genetics , 3T3 Cells , Animals , Base Sequence , Exons/genetics , Homeobox Protein Nkx-2.2 , Humans , Islets of Langerhans/cytology , Islets of Langerhans/embryology , Mice , Molecular Sequence Data , Nuclear Proteins , Promoter Regions, Genetic/genetics , Zebrafish Proteins
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