ABSTRACT
BACKGROUND: Nonsteroidal anti-inflammatory drugs-exacerbated respiratory disease (N-ERD) is currently classified as a type-2 (T2) immune-mediated disease characterized by asthma, chronic rhinosinusitis, and hypersensitivity to cyclooxygenase-1 inhibitors. OBJECTIVES: The aim of this study was to characterize immunological endotypes of N-ERD based on the gene expression profile in the bronchial epithelium. METHODS: mRNA transcriptome (mRNA-sequencing) was analyzed in bronchial brushings from patients with N-ERD (n = 22), those with nonsteroidal anti-inflammatory drug-tolerant asthma (NTA, n = 21), and control subjects (n = 11). Additionally, lipid and protein mediators were measured in bronchoalveolar lavage fluid (BALF). RESULTS: Initial analysis of the entire asthma group revealed 2 distinct gene expression signatures: "T2-high" with increased expression of T2-related genes (eg, CLCA1, CST1), and "proinflammatory" characterized by the expression of innate immunity (eg, FOSB, EGR3) and IL-17A response genes. These endotypes showed similar prevalence in N-ERD and NTA (eg, T2-high: 33% and 32%, respectively). T2-high asthma was characterized by increased expression of mast cell and eosinophil markers, goblet cell hyperplasia, and elevated LTE4 and PGD2 in BALF. Patients with a proinflammatory endotype showed mainly neutrophilic inflammation and increased innate immunity mediators in BALF. Furthermore, the proinflammatory signature was associated with a more severe course of asthma and marked airway obstruction. These signatures could be recreated in vitro by exposure of bronchial epithelial cells to IL-13 (T2-high) and IL-17A (proinflammatory). CONCLUSIONS: T2-high signature was found only in one-third of patients with N-ERD, which was similar to what was found in patients with NTA. The proinflammatory endotype, which also occurred in N-ERD, suggests a novel mechanism of severe disease developing on a non-T2 background.
Subject(s)
Asthma , Respiration Disorders , Respiratory Tract Diseases , Humans , Transcriptome , Interleukin-17/genetics , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Asthma/genetics , Epithelial CellsABSTRACT
BACKGROUND: miR-10a-5p has been shown to regulate cancer cell proliferation and invasiveness and endothelial cell inflammatory responses. The function of miR-10a-5p in the skin has not been previously studied. The aim of the current study was to examine miR-10a-5p expression, regulation, and function in keratinocytes (KCs) in association with atopic dermatitis (AD). METHODS: The expression of miR-10a-5p and its target genes was analyzed using RT-qPCR, mRNA array analysis, in situ hybridization, and immunofluorescence. The transfection of miRNA mimics, cell cycle distribution analysis, and luciferase assays was used to study miR-10a-5p functions in human primary KCs. RESULTS: miR-10a-5p was found to be upregulated in lesional skin from patients with AD and in proliferating KCs. Array and pathway analysis of IL-1ß-stimulated KCs revealed that miR-10a-5p inhibited many genes that affect cell cycle progression and only a few inflammation-related genes. Accordingly, fewer cells in S-phase and reduced proliferation were detected as characteristics of miR-10a-5p-transfected KCs. The influence of miR-10a-5p on cell proliferation was also evident in KCs induced by AD-related cytokines, including IL-4, IL-17, and IL-1ß, as measured by the capacity to strongly suppress the expression of the proliferation marker Ki-67. Among AD-related putative direct target genes, we verified hyaluronan synthase 3, a damage-associated positive regulator of KC migration and proliferation, as a direct target of miR-10a-5p. CONCLUSIONS: miR-10a-5p inhibits KC proliferation and directly targets hyaluronan synthase 3 and thereby may modulate AD-associated processes in the skin.
Subject(s)
Dermatitis, Atopic/etiology , Dermatitis, Atopic/metabolism , Gene Expression Regulation , Keratinocytes/metabolism , MicroRNAs/genetics , RNA Interference , Adult , Cell Cycle/genetics , Cell Differentiation/genetics , Cell Proliferation/genetics , Cytokines/metabolism , Dermatitis, Atopic/pathology , Disease Susceptibility , Female , Gene Expression Profiling , Humans , Male , Middle Aged , Skin/immunology , Skin/metabolism , Skin/pathology , Young AdultABSTRACT
There have been extensive developments on cellular and molecular mechanisms of immune regulation in allergy, asthma, autoimmune diseases, tumor development, organ transplantation, and chronic infections during the last few years. Better understanding the functions, reciprocal regulation, and counterbalance of subsets of immune and inflammatory cells that interact through interleukins, interferons, TNF-α, and TGF-ß offer opportunities for immune interventions and novel treatment modalities in the era of development of biological immune response modifiers particularly targeting these molecules or their receptors. More than 60 cytokines have been designated as interleukins since the initial discoveries of monocyte and lymphocyte interleukins (called IL-1 and IL-2, respectively). Studies of transgenic or gene-deficient mice with altered expression of these cytokines or their receptors and analyses of mutations and polymorphisms in human genes that encode these products have provided essential information about their functions. Here we review recent developments on IL-1 to IL-38, TNF-α, TGF-ß, and interferons. We highlight recent advances during the last few years in this area and extensively discuss their cellular sources, targets, receptors, signaling pathways, and roles in immune regulation in patients with allergy and asthma and other inflammatory diseases.
Subject(s)
Immune System Diseases , Interferons/physiology , Interleukins/physiology , T-Lymphocytes, Regulatory/immunology , Transforming Growth Factor beta/physiology , Tumor Necrosis Factor-alpha/physiology , Animals , HumansABSTRACT
Little is known about the functions of microRNAs (miRNAs) in skin pigmentation disorders. The aim of this study was to investigate the expression and potential role of miRNAs in vitiligo. Of 12 studied miRNAs with proven functions in cell proliferation, differentiation, immune responses and melanogenesis, miR-99b, miR-125b, miR-155 and miR-199a-3p were found to be increased and miR-145 was found to be decreased in the skin of patients with vitiligo. Combined pathway and target analysis revealed melanogenesis-associated targets for miR-99b, miR-125b, miR-155 and miR-199a-3p. In situ hybridization analysis demonstrated increased expression of miR-155 in the epidermis of patients with vitiligo. Correspondingly, miR-155 was induced by vitiligo-associated cytokines in human primary melanocytes and keratinocytes. When overexpressed, miR-155 inhibited the expression of melanogenesis-associated genes and altered interferon-regulated genes in melanocytes and keratinocytes. In conclusion, this study demonstrates that the expression of miRNAs is dysregulated in the skin of patients with vitiligo and suggests that miR-155 contributes to the pathogenesis of vitiligo.
Subject(s)
Keratinocytes/metabolism , Melanocytes/metabolism , MicroRNAs/metabolism , Vitiligo/metabolism , Case-Control Studies , Cell Differentiation , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Humans , In Situ Hybridization , Real-Time Polymerase Chain Reaction , Vitiligo/pathologyABSTRACT
BACKGROUND: Chronic skin inflammation in atopic dermatitis (AD) is associated with elevated expression of proinflammatory genes and activation of innate immune responses in keratinocytes. microRNAs (miRNAs) are short, single-stranded RNA molecules that silence genes via the degradation of target mRNAs or inhibition of translation. OBJECTIVE: The aim of this study was to investigate the role of miR-146a in skin inflammation in AD. METHODS: RNA and protein expression was analyzed using miRNA and mRNA arrays, RT-quantitative PCR, Western blotting, and immunonohistochemistry. Transfection of miR-146a precursors and inhibitors into human primary keratinocytes, luciferase assays, and MC903-dependent mouse model of AD were used to study miR-146a function. RESULTS: We show that miR-146a expression is increased in keratinocytes and chronic lesional skin of patients with AD. miR-146a inhibited the expression of numerous proinflammatory factors, including IFN-γ-inducible and AD-associated genes CCL5, CCL8, and ubiquitin D (UBD) in human primary keratinocytes stimulated with IFN-γ, TNF-α, or IL-1ß. In a mouse model of AD, miR-146a-deficient mice developed stronger inflammation characterized by increased accumulation of infiltrating cells in the dermis, elevated expression of IFN-γ, CCL5, CCL8, and UBD in the skin, and IFN-γ, IL-1ß, and UBD in draining lymph nodes. Both tissue culture and in vivo experiments in mice demonstrated that miR-146a-mediated suppression in allergic skin inflammation partially occurs through direct targeting of upstream nuclear factor kappa B signal transducers caspase recruitment domain-containing protein 10 and IL-1 receptor-associated kinase 1. In addition, human CCL5 was determined as a novel, direct target of miR-146a. CONCLUSION: Our data demonstrate that miR-146a controls nuclear factor kappa B-dependent inflammatory responses in keratinocytes and chronic skin inflammation in AD.
Subject(s)
Dermatitis, Atopic/genetics , Keratinocytes/immunology , MicroRNAs/physiology , NF-kappa B/metabolism , RNA Interference , Skin/immunology , Animals , Calcitriol/administration & dosage , Calcitriol/analogs & derivatives , Cell Movement/genetics , Cells, Cultured , Chronic Disease , Cytokines/genetics , Cytokines/immunology , Cytokines/metabolism , Dermatitis, Atopic/chemically induced , Dermatitis, Atopic/immunology , Disease Models, Animal , Humans , Immunity, Innate , Immunosuppression Therapy , Inflammation/genetics , Inflammation Mediators/metabolism , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , NF-kappa B/genetics , RNA Interference/immunology , Signal Transduction/genetics , Skin/pathology , Up-RegulationABSTRACT
Human rhinoviruses (RVs) are a major cause of exacerbations in asthma and other chronic airway diseases. A characteristic feature of asthmatic epithelium is goblet cell metaplasia and mucus hypersecretion. Bronchial epithelium is also an important source of lipid mediators, including pro- and antiinflammatory eicosanoids. By using air-liquid interface cultures of airway epithelium from patients with asthma and nonasthmatic control subjects, we compared RV16 replication-induced changes in mRNA expression of asthma candidate genes and eicosanoid production in the epithelium with or without IL-13-induced mucus metaplasia. Mucus metaplastic epithelium was characterized by a 20-fold less effective replication of RV16 and blunted changes in gene expression; this effect was seen to the same extent in patients with asthma and control subjects. We identified ciliary cells as the main target for RV16 by immunofluorescence imaging and demonstrated that the numbers of ciliary cells decreased in RV16-infected epithelium. RV16 infection of mucociliary epithelium resulted in overexpression of genes associated with bronchial remodeling (e.g., MUC5AC, FGF2, and HBEGF), induction of cyclooxygenase-2, and increased secretion of prostaglandins. These responses were similar in both studied groups. These data indicate that structural changes associated with mucus metaplasia renders airway epithelium less susceptible to RV infection. Thus, exacerbations of the lung disease caused by RV may result from severe impairment in mucociliary clearance or activation of immune defense rather than from preferential infection of mucus metaplastic epithelium. Repeated rhinoviral infections of compromised epithelium may contribute to the remodeling of the airways.
Subject(s)
Asthma/immunology , Bronchi/immunology , Cytokines/metabolism , Epithelial Cells/immunology , Mucus/metabolism , Picornaviridae Infections/prevention & control , Rhinovirus/immunology , Th2 Cells/immunology , Adult , Airway Remodeling , Asthma/genetics , Asthma/pathology , Bronchi/pathology , Bronchi/virology , Case-Control Studies , Cells, Cultured , Disease Susceptibility , Epithelial Cells/pathology , Epithelial Cells/virology , Female , Gene Expression Profiling , Gene Expression Regulation , Humans , Intercellular Adhesion Molecule-1/metabolism , Male , Metaplasia , Middle Aged , Mucociliary Clearance , Picornaviridae Infections/genetics , Picornaviridae Infections/immunology , Picornaviridae Infections/pathology , Picornaviridae Infections/virology , Prostaglandins/metabolism , RNA, Messenger/metabolism , Rhinovirus/growth & development , Rhinovirus/pathogenicity , Th2 Cells/virology , Time Factors , Virus ReplicationABSTRACT
BACKGROUND: Enhanced apoptosis of keratinocytes is the main cause of eczema and spongiosis in patients with the common inflammatory skin disease atopic dermatitis (AD). OBJECTIVE: The aim of the study was to investigate molecular mechanisms of AD-related apoptosis of keratinocytes. METHODS: Primary keratinocytes isolated from patients with AD and healthy donors were used to study apoptosis by using annexin V/7-aminoactinomycin D staining. Illumina mRNA Expression BeadChips, quantitative RT-PCR, and immunofluorescence were used to study gene expression. In silico analysis of candidate genes was performed on genome-wide single nucleotide polymorphism data. RESULTS: We demonstrate that keratinocytes of patients with AD exhibit increased IFN-γ-induced apoptosis compared with keratinocytes from healthy subjects. Further mRNA expression analyses revealed differential expression of apoptosis-related genes in AD keratinocytes and skin and the upregulation of immune system-related genes in skin biopsy specimens of chronic AD lesions. Three apoptosis-related genes (NOD2, DUSP1, and ADM) and 8 genes overexpressed in AD skin lesions (CCDC109B, CCL5, CCL8, IFI35, LYN, RAB31, IFITM1, and IFITM2) were induced by IFN-γ in primary keratinocytes. The protein expression of IFITM1, CCL5, and CCL8 was verified in AD skin. In line with the functional studies and AD-related mRNA expression changes, in silico analysis of genome-wide single nucleotide polymorphism data revealed evidence of an association between AD and genetic markers close to or within the IFITM cluster or RAB31, DUSP1, and ADM genes. CONCLUSION: Our results demonstrate increased IFN-γ responses in skin of patients with AD and suggest involvement of multiple new apoptosis- and inflammation-related factors in the development of AD.
Subject(s)
Apoptosis/immunology , Dermatitis, Atopic/immunology , Interferon-gamma/immunology , Keratinocytes/immunology , Skin/pathology , Adrenomedullin/genetics , Adrenomedullin/immunology , Adrenomedullin/metabolism , Aged , Antigens, Differentiation/genetics , Antigens, Differentiation/immunology , Antigens, Differentiation/metabolism , Apoptosis/drug effects , Biopsy , Cells, Cultured , Chemokine CCL5/genetics , Chemokine CCL5/immunology , Chemokine CCL5/metabolism , Chemokine CCL8/genetics , Chemokine CCL8/immunology , Chemokine CCL8/metabolism , Computational Biology , Dermatitis, Atopic/genetics , Dermatitis, Atopic/metabolism , Dual Specificity Phosphatase 1/genetics , Dual Specificity Phosphatase 1/immunology , Dual Specificity Phosphatase 1/metabolism , Female , Gene Expression Profiling , Genetic Markers/genetics , Genome-Wide Association Study , Humans , Interferon-gamma/pharmacology , Keratinocytes/drug effects , Keratinocytes/pathology , Male , Middle Aged , Nod2 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/immunology , Nod2 Signaling Adaptor Protein/metabolism , Polymorphism, Single Nucleotide , Up-Regulation/immunologyABSTRACT
BACKGROUND: Acute myeloid leukemia (AML) patients have limited effect from T-cell-based therapies, such as PD-1 and CTLA-4 blockade. However, recent data indicate that AML patients with TP53 mutation have higher immune infiltration and other immunomodulatory therapies could thus potentially be effective. Here, we performed the transcriptional analysis of distinct T-cell subpopulations from TP53-mutated AML to identify gene expression signatures suggestive of altered functional properties. METHODS: CD8+ cytotoxic T lymphocytes (CTLs), conventional helper T cells (Th), and regulatory T cells (Tregs) were sorted from peripheral blood of AML patients with TP53 mutation (n = 5) and healthy donors (n = 3), using FACS, and the different subpopulations were subsequently subjected to RNA-sequencing. Differentially expressed genes were identified and gene set enrichment analysis (GSEA) was performed to outline altered pathways and exhaustion status. Also, expression levels for a set of genes encoding established and emerging immuno-oncological targets were defined. RESULTS: The results showed altered transcriptional profiles for each of the T-cell subpopulations from TP53-mutated AML as compared to control subjects. IFN-α and IFN-γ signaling were stronger in TP53-mutated AML for both CTLs and Tregs. Furthermore, in TP53-mutated AML as compared to healthy controls, Tregs showed gene expression signatures suggestive of metabolic adaptation to their environment, whereas CTLs exhibited features of exhaustion/dysfunction with a stronger expression of TIM3 as well as enrichment of a gene set related to exhaustion. CONCLUSIONS: The results provide insights on mechanisms underlying the inadequate immune response to leukemic cells in TP53-mutated AML and open up for further exploration toward novel treatment regimens for these patients.
Subject(s)
Leukemia, Myeloid, Acute , T-Lymphocytes, Regulatory , CD8-Positive T-Lymphocytes , Humans , Leukemia, Myeloid, Acute/metabolism , Mutation , T-Lymphocytes, Cytotoxic , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Rhinovirus (RV) infections are associated with asthma exacerbations. MicroRNA-146a and microRNA-146b (miR-146a/b) are anti-inflammatory miRNAs that suppress signaling through the nuclear factor kappa B (NF-κB) pathway and inhibit pro-inflammatory chemokine production in primary human bronchial epithelial cells (HBECs). In the current study, we aimed to explore whether miR-146a/b could regulate cellular responses to RVs in HBECs and airways during RV-induced asthma exacerbation. We demonstrated that expression of miR-146a/b and pro-inflammatory chemokines was increased in HBECs and mouse airways during RV infection. However, transfection with cell-penetrating peptide (CPP)-miR-146a nanocomplexes before infection with RV significantly reduced the expression of the pro-inflammatory chemokines CCL5, IL-8 and CXCL1, increased interferon-λ production, and attenuated infection with the green fluorescent protein (GFP)-expressing RV-A16 in HBECs. Concordantly, compared to wild-type (wt) mice, Mir146a/b-/- mice exhibited more severe airway neutrophilia and increased T helper (Th)1 and Th17 cell infiltration in response to RV-A1b infection and a stronger Th17 response with a less prominent Th2 response in house dust mite extract (HDM)-induced allergic airway inflammation and RV-induced exacerbation models. Interestingly, intranasal administration of CPP-miR-146a nanocomplexes reduced HDM-induced allergic airway inflammation without a significant effect on the Th2/Th1/Th17 balance in wild-type mice. In conclusion, the overexpression of miR-146a has a strong anti-inflammatory effect on RV infection in HBECs and a mouse model of allergic airway inflammation, while a lack of miR-146a/b leads to attenuated type 2 cell responses in mouse models of allergic airway inflammation and RV-induced exacerbation of allergic airway inflammation. Furthermore, our data indicate that the application of CPP-miR-146a nanocomplexes has therapeutic potential for targeting airway inflammation.
Subject(s)
Asthma/pathology , Hypersensitivity/pathology , Inflammation/pathology , MicroRNAs/genetics , Picornaviridae Infections/complications , Th2 Cells/immunology , Adult , Allergens , Animals , Asthma/etiology , Asthma/metabolism , Disease Models, Animal , Female , Humans , Hypersensitivity/etiology , Hypersensitivity/metabolism , Inflammation/etiology , Inflammation/metabolism , Male , Mice , Picornaviridae Infections/virology , Rhinovirus/physiologyABSTRACT
BACKGROUND: The role of miRNAs in the pathogenesis and determining the phenotypes of asthma is not fully elucidated. miR-146a has been previously shown to suppress inflammatory responses in different cells. In this study, we investigated the functions of miR-146a in human bronchial epithelial cells (HBECs) in association with neutrophilic, eosinophilic, and paucigranulocytic phenotypes of asthma. METHODS: Bronchial brushing specimens and brochial mucosal biopsy samples were collected from adult patients with asthma and from age- and gender-matched non-asthmatic individuals. The expression of miR-146a in bronchial brushing specimens, bronchial biopsy tissue sections or cultured primary bronchial epithelial cells was analyzed by RT-qPCR or by in situ hybridization. The expression of direct and indirect miR-146a target genes was determined by RT-qPCR or ELISA. The migration of neutrophils was studied by neutrophil chemotaxis assay and flow cytometry. For statistical analysis, unpaired two-way Student's t test, one-way ANOVA or linear regression analysis were used. RESULTS: Reduced expression of miR-146a was found in bronchial brushing specimens from asthma patients as compared to non-asthmatics and irrespective of the phenotype of asthma. In the same samples, the neutrophil attracting chemokines IL-8 and CXCL1 showed increased expression in patients with neutrophilic asthma and increased IL-33 expression was found in patients with eosinophilic asthma. Linear regression analysis revealed a significant negative association between the expression of miR-146a in bronchial brushings and neutrophil cell counts in bronchoalveolar lavage fluid of patients with asthma. In bronchial biopsy specimens, the level of miR-146a was highest in the epithelium as determined with in situ hybridization. In primary conventional HBEC culture, the expression of miR-146a was induced in response to the stimulation with IL-17A, TNF-α, and IL-4. The mRNA expression and secretion of IL-8 and CXCL1 was inhibited in both stimulated and unstimulated HBECs transfected with miR-146a mimics. Supernatants from HBECs transfected with miR-146a had reduced capability of supporting neutrophil migration in neutrophil chemotaxis assay. CONCLUSION: Our results suggest that decreased level of miR-146a in HBECs from patients with asthma may contribute to the development of neutrophilic phenotype of asthma.
ABSTRACT
BACKGROUND: Microalbuminuria (MA) is recognized as an important risk factor for cardiovascular and renal complications in diabetes. We sought to evaluate how screening for MA is conducted and how urine albumin (UA) results are interpreted in primary care internationally. METHODS: General practitioners (GPs) received a case history-based questionnaire depicting a male type 2 diabetes patient in whom UA testing had not been performed. Questions were related to type of urine sample used for UA testing, need for a repeat test, whether UA testing was performed in the office laboratory, and what changes in UA results were considered clinically important [critical difference (CD)]. Participants received national benchmarking feedback reports. RESULTS: We included 2078 GPs from 9 European countries. Spot urine samples were used most commonly for first time office-based testing, whereas timed collections were used to a larger extent for hospital-based repeat tests. Repeat tests were requested by 45%-77% of GPs if the first test was positive. Four different measurement units were used by 70% of participants in estimating clinically important changes in albumin values. Stated CDs varied considerably among GPs, with similar variations in each country. A median CD of 33% was considered clinically important for both improvement and deterioration in MA, corresponding to an achievable analytical imprecision of 14%, when UA is reported as an albumin/creatinine ratio. CONCLUSIONS: Guidelines on diagnosing MA are followed only partially, and should be made more practicable, addressing issues such as type of samples, measurement units, and repeat tests.
Subject(s)
Albuminuria/urine , Primary Health Care , Quality Control , Australia , Diabetes Mellitus, Type 2/urine , Europe , Humans , Internationality , Specimen Handling , Surveys and QuestionnairesABSTRACT
BACKGROUND: Tonsils provide an innovative in vivo model for investigating immune response to infections and allergens. However, data are scarce on the differences in tonsillar virus infections and immune responses between patients with tonsillar hypertrophy or recurrent tonsillitis. We investigated the differences in virus detection and T cell and interferon gene expression in patients undergoing tonsillectomy due to tonsillar hypertrophy or recurrent tonsillitis. METHODS: Tonsils of 89 surgical patients with tonsillar hypertrophy (n = 47) or recurrent tonsillitis (n = 42) were analysed. Patients were carefully characterized clinically. Standard questionnaire was used to asses preceding and allergy symptoms. Respiratory viruses were analysed in tonsils and nasopharynx by PCR. Quantitative real-time PCR was used to analyse intratonsillar gene expressions of IFN-α, IFN-ß, IFN-γ, IL-10, IL-13, IL-17, IL-28, IL-29, IL-37, TGF-ß, FOXP3, GATA3, RORC2 and Tbet. RESULTS: Median age of the subjects was 15 years (range 2-60). Patients with tonsillar hypertrophy were younger, smoked less often, had less pollen allergy and had more adenovirus, bocavirus-1, coronavirus and rhinovirus in nasopharynx (all P < 0.05). Only bocavirus-1 was more often detected in hypertrophic tonsils (P < 0.05). In age-adjusted analysis, tonsillar hypertrophy was associated with higher mRNA expressions of IL-37 (P < 0.05). CONCLUSIONS: Intratonsillar T cell and interferon gene expressions appeared to be relatively stable for both tonsillar hypertrophy and recurrent tonsillitis. Of the studied cytokines, only newly discovered anti-inflammatory cytokine IL-37, was independently associated with tonsillar hypertrophy showing slightly stronger anti-inflammatory response in these patients.
ABSTRACT
BACKGROUND: Spliceosomal snRNAs and ribosomal RNAs in metazoans contain numerous modified residues that are functionally important. The most common modifications are site-specific 2'-O-methylation and pseudouridylation, both directed by small ribonucleoprotein particles. Each particle is composed of a short guide RNA and a set of several proteins. All previously characterized modification guide RNAs in metazoa are encoded in and processed from introns. RESULTS: We have identified and characterized three novel guide RNAs for conserved 2'-O-methylation of U2, U4, and U12 snRNAs. Two guides, termed mgU2-25/61 and mgU12-22/U4-8, appear to be independently transcribed as judged by the presence of methylated guanosine caps at their 5' ends and upstream promoters similar to those of telomerase RNA. These guide RNAs are each composed of a canonical box C/D snoRNA and a novel box C/D snoRNA-like domain, where the C'/D' motif, rather than C/D, can be folded into a conserved kink-turn structure. The snoRNA-like domains are predicted to direct 2'-O-methylation of invariant G residues that occupy analogous positions in the U2 and U12 snRNA secondary structures. A third guide, mgU2-19/30 RNA, is composed of two canonical box C/D snoRNA domains encoded within a single intron. CONCLUSIONS: This is the first description in metazoan cells of 5'-capped modification guide RNAs that appear to be independently transcribed. Since plant, yeast, and protozoan guide RNAs are mostly independently transcribed, the identification of such RNAs argues that ancestral metazoans possessed independently transcribed guide RNAs and only later, during the evolution of metazoan organisms, did the guide RNA genes shift to introns.
Subject(s)
RNA Processing, Post-Transcriptional , RNA, Small Nucleolar , RNA, Small Untranslated , Spliceosomes , Vertebrates , Animals , Humans , Base Pairing , Base Sequence , Blotting, Northern , Cell Fractionation , DNA Methylation , Immunoprecipitation , Introns/genetics , Molecular Sequence Data , Oligonucleotides , RNA Processing, Post-Transcriptional/genetics , RNA, Small Nucleolar/genetics , RNA, Small Nucleolar/metabolism , Sequence Alignment , Spliceosomes/genetics , Vertebrates/genetics , RNA, Small Untranslated/geneticsABSTRACT
miR-146a inhibits inflammatory responses in human keratinocytes and in different mouse models of skin inflammation. Little is known about the role of miR-146b in the skin. In this study, we confirmed the increased expression of miR-146a and miR-146b (miR-146a/b) in the lesional skin of patients with psoriasis. The expression of miR-146a was approximately twofold higher than that of miR-146b in healthy human skin, and it was more strongly induced by stimulation of proinflammatory cytokines in keratinocytes and fibroblasts. miR-146a/b target genes regulating inflammatory responses or proliferation were altered in the skin of patients with psoriasis, among which FERMT1 was verified as a direct target of miR-146a. In silico analysis of genome-wide data from >4,000 psoriasis cases and >8,000 controls confirmed a moderate association between psoriasis and genetic variants in the miR-146a encoding gene. Transfection of miR-146a/b suppressed and inhibition enhanced keratinocyte proliferation and the expression of psoriasis-related target genes. Enhanced expression of miR-146a/b-influenced genes was detected in cultured keratinocytes from miR-146a-/- and skin fibroblasts from miR-146a-/- and miR-146b-/- mice stimulated with psoriasis-associated cytokines as compared with wild-type mice. Our results indicate that besides miR-146a, miR-146b is expressed and might be capable of modulation of inflammatory responses and keratinocyte proliferation in psoriatic skin.
Subject(s)
Cell Proliferation/genetics , Gene Expression Regulation , Keratinocytes/metabolism , MicroRNAs/genetics , Psoriasis/genetics , Animals , Apoptosis/genetics , Case-Control Studies , Cells, Cultured , Dermatitis/genetics , Dermatitis/pathology , Disease Models, Animal , Humans , Mice , Mice, Inbred C57BL , Psoriasis/pathology , Real-Time Polymerase Chain Reaction/methodsABSTRACT
AIMS: To assess general practitioners (GPs) knowledge of guideline recommendations on diagnosing microalbuminuria (MA) and to evaluate how this diagnosis influences drug treatment of diabetes patients. METHODS: A postal case-history based questionnaire describing a male patient (previously not tested for MA) with type 2 diabetes who had several risk markers for cardiovascular disease. RESULTS: 2078GPs from nine European countries were included, with response rates varying from 7% to 43%. Almost all GPs recommended annual testing for MA. Forty-five to 77% (depending on country) of GPs required more than one positive test to diagnose MA. The absolute increase in the percentages of GPs who would supplement the patient's drug treatment if MA developed was: for anginotensin converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs) 23-50% (depending on country), for statins 0-19%, for acetylsalicylic acid 2-13%, and for hypoglycemic agents (tablets and insulin) 0-33%. The proportion of GPs recommending all four possible treatment modalities was low. CONCLUSIONS: Guidelines for diagnosing MA were partly followed. ACEIs and ARBs were recommended when MA was present, but the recommended multifactorial treatment of cardiovascular risk markers was not implemented.
Subject(s)
Albuminuria/diagnosis , Albuminuria/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Data Collection , Diabetes Mellitus, Type 2/complications , Humans , Male , Middle Aged , Surveys and QuestionnairesABSTRACT
Several mRNA-binding proteins, including hnRNP A1 and HuR, contain bidirectional transport signals that mediate both their nuclear import and export. Previously, Transportin 1 (Trn1) was identified as a mediator of hnRNP A1 import, whereas the closely related protein Transportin 2 (Trn2) was shown to interact with HuR. Here we have investigated the subfamily of transportins that consists of Trn1 (or Kap beta2A) and two alternatively spliced Trn2 isoforms (Trn2a and Trn2b), also called Trn2 and Kap beta2B. The sequence differences among these proteins could alter either their cargo specificity or their response to RanGTP and thus their function as import or export receptors. Using in vitro binding assays, we show that hnRNP A1 preferentially binds Trn1 and Trn2b versus Trn2a. HuR interacts with all three transportins, as well as weakly with Imp beta. The hnRNP A1 and HuR shuttling domains, called M9 and HNS, respectively, are sufficient for these interactions. Despite small differences in the binding of HuR and hnRNP A1 to the three transportins, in vitro interaction studies performed in the presence and absence of RanQ69LGTP indicate that all three transportins most likely act as import factors for HuR and hnRNP A1. In digitonin-permeabilized HeLa cells, both M9 and HNS peptides compete for the import of recombinant hnRNP A1 and HuR, indicating that HuR and hnRNP A1 import pathways are at least partially overlapping. Possible nucleocytoplasmic shuttling mechanisms for hnRNP A1 and HuR are discussed.