ABSTRACT
Genome-wide association studies have uncovered thousands of common variants associated with human disease, but the contribution of rare variants to common disease remains relatively unexplored. The UK Biobank contains detailed phenotypic data linked to medical records for approximately 500,000 participants, offering an unprecedented opportunity to evaluate the effect of rare variation on a broad collection of traits1,2. Here we study the relationships between rare protein-coding variants and 17,361 binary and 1,419 quantitative phenotypes using exome sequencing data from 269,171 UK Biobank participants of European ancestry. Gene-based collapsing analyses revealed 1,703 statistically significant gene-phenotype associations for binary traits, with a median odds ratio of 12.4. Furthermore, 83% of these associations were undetectable via single-variant association tests, emphasizing the power of gene-based collapsing analysis in the setting of high allelic heterogeneity. Gene-phenotype associations were also significantly enriched for loss-of-function-mediated traits and approved drug targets. Finally, we performed ancestry-specific and pan-ancestry collapsing analyses using exome sequencing data from 11,933 UK Biobank participants of African, East Asian or South Asian ancestry. Our results highlight a significant contribution of rare variants to common disease. Summary statistics are publicly available through an interactive portal ( http://azphewas.com/ ).
Subject(s)
Biological Specimen Banks , Databases, Genetic , Disease/genetics , Exome/genetics , Genetic Variation/genetics , Adult , Aged , Female , Genome-Wide Association Study , Humans , Male , Middle Aged , Phenotype , Proteins/chemistry , Proteins/genetics , United Kingdom , Exome SequencingABSTRACT
BACKGROUND: Chronic obstructive pulmonary disease (COPD) patients are at increased risk of poor outcome from Coronavirus disease (COVID-19). Early data suggest elevated Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) receptor angiotensin converting enzyme 2 (ACE2) expression, but relationships to disease phenotype and downstream regulators of inflammation in the Renin-Angiotensin system (RAS) are unknown. We aimed to determine the relationship between RAS gene expression relevant to SARS-CoV-2 infection in the lung with disease characteristics in COPD, and the regulation of newly identified SARS-CoV-2 receptors and spike-cleaving proteases, important for SARS-CoV-2 infection. METHODS: We quantified gene expression using RNA sequencing of epithelial brushings and bronchial biopsies from 31 COPD and 37 control subjects. RESULTS: ACE2 gene expression (log2-fold change (FC)) was increased in COPD compared to ex-smoking (HV-ES) controls in epithelial brushings (0.25, p = 0.042) and bronchial biopsies (0.23, p = 0.050), and correlated with worse lung function (r = - 0.28, p = 0.0090). ACE2 was further increased in frequent exacerbators compared to infrequent exacerbators (0.51, p = 0.00045) and associated with use of ACE inhibitors (ACEi) (0.50, p = 0.0034), having cardiovascular disease (0.23, p = 0.048) or hypertension (0.34, p = 0.0089), and inhaled corticosteroid use in COPD subjects in bronchial biopsies (0.33, p = 0.049). Angiotensin II receptor type (AGTR)1 and 2 expression was decreased in COPD bronchial biopsies compared to HV-ES controls with log2FC of -0.26 (p = 0.033) and - 0.40, (p = 0.0010), respectively. However, the AGTR1:2 ratio was increased in COPD subjects compared with HV-ES controls, log2FC of 0.57 (p = 0.0051). Basigin, a newly identified potential SARS-CoV-2 receptor was also upregulated in both brushes, log2FC of 0.17 (p = 0.0040), and bronchial biopsies, (log2FC of 0.18 (p = 0.017), in COPD vs HV-ES. Transmembrane protease, serine (TMPRSS)2 was not differentially regulated between control and COPD. However, various other spike-cleaving proteases were, including TMPRSS4 and Cathepsin B, in both epithelial brushes (log2FC of 0.25 (p = 0.0012) and log2FC of 0.56 (p = 5.49E-06), respectively) and bronchial biopsies (log2FC of 0.49 (p = 0.00021) and log2FC of 0.246 (p = 0.028), respectively). CONCLUSION: This study identifies key differences in expression of genes related to susceptibility and aetiology of COVID-19 within the COPD lung. Further studies to understand the impact on clinical course of disease are now required.
Subject(s)
COVID-19/genetics , Lung/metabolism , Pulmonary Disease, Chronic Obstructive/genetics , Transcriptome , Aged , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Basigin/genetics , Basigin/metabolism , COVID-19/diagnosis , COVID-19/metabolism , COVID-19/physiopathology , Case-Control Studies , Female , Forced Expiratory Volume , Gene Expression Regulation , Humans , Lung/physiopathology , Male , Middle Aged , Prognosis , Pulmonary Disease, Chronic Obstructive/diagnosis , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/physiopathology , Receptor, Angiotensin, Type 1/genetics , Receptor, Angiotensin, Type 1/metabolism , Receptor, Angiotensin, Type 2/genetics , Receptor, Angiotensin, Type 2/metabolism , Vital CapacityABSTRACT
Rationale: Viral infections are major drivers of exacerbations and clinical burden in patients with asthma and chronic obstructive pulmonary disease (COPD). IFN-ß is a key component of the innate immune response to viral infection. To date, studies of inhaled IFN-ß treatment have not demonstrated a significant effect on asthma exacerbations.Objectives: The dynamics of exogenous IFN-ß activity were investigated to inform on future clinical indications for this potential antiviral therapy.Methods: Monocyte-derived macrophages (MDMs), alveolar macrophages, and primary bronchial epithelial cells (PBECs) were isolated from healthy control subjects and patients with COPD and infected with influenza virus either prior to or after IFN-ß stimulation. Infection levels were measured by the percentage of nucleoprotein 1-positive cells using flow cytometry. Viral RNA shedding and IFN-stimulated gene expression were measured by quantitative PCR. Production of inflammatory cytokines was measured using MSD.Measurements and Main Results: Adding IFN-ß to MDMs, alveolar macrophages, and PBECs prior to, but not after, infection reduced the percentage of nucleoprotein 1-positive cells by 85, 56, and 66%, respectively (P < 0.05). Inhibition of infection lasted for 24 hours after removal of IFN-ß and was maintained albeit reduced up to 1 week in MDMs and 72 hours in PBECs; this was similar between healthy control subjects and patients with COPD. IFN-ß did not induce inflammatory cytokine production by MDMs or PBECs but reduced influenza-induced IL-1ß production by PBECs.Conclusions:In vitro modeling of IFN-ß dynamics highlights the potential for intermittent prophylactic doses of exogenous IFN-ß to modulate viral infection. This provides important insights to aid the future design of clinical trials of IFN-ß in asthma and COPD.
Subject(s)
Antiviral Agents/therapeutic use , Asthma/drug therapy , Interferon-beta/therapeutic use , Pulmonary Disease, Chronic Obstructive/drug therapy , Virus Diseases/drug therapy , Adult , Aged , Aged, 80 and over , Asthma/immunology , Asthma/virology , Female , Humans , Male , Middle Aged , Pulmonary Disease, Chronic Obstructive/immunology , Pulmonary Disease, Chronic Obstructive/virology , Virus Diseases/immunologyABSTRACT
BACKGROUND: Immune repertoire sequencing of the T-cell receptor can identify clonotypes that have expanded as a result of antigen recognition or hematological malignancies. However, current sequencing protocols display limitations with nonuniform amplification and polymerase-induced errors during sequencing. Here, we developed a sequencing method that overcame these issues and applied it to γδ T cells, a cell type that plays a unique role in immunity, autoimmunity, homeostasis of intestine, skin, adipose tissue, and cancer biology. METHODS: The ultrasensitive immune repertoire sequencing method used PCR-introduced unique molecular identifiers. We constructed a 32-panel assay that captured the full diversity of the recombined T-cell receptor delta loci in γδ T cells. The protocol was validated on synthetic reference molecules and blood samples of healthy individuals. RESULTS: The 32-panel assay displayed wide dynamic range, high reproducibility, and analytical sensitivity with single-nucleotide resolution. The method corrected for sequencing-depended quantification bias and polymerase-induced errors and could be applied to both enriched and nonenriched cells. Healthy donors displayed oligoclonal expansion of γδ T cells and similar frequencies of clonotypes were detected in both enrichment and nonenriched samples. CONCLUSIONS: Ultrasensitive immune repertoire sequencing strategy enables quantification of individual and specific clonotypes in a background that can be applied to clinical as well as basic application areas. Our approach is simple, flexible, and can easily be implemented in any molecular laboratory.
Subject(s)
DNA/analysis , Intraepithelial Lymphocytes/classification , Base Sequence , DNA/genetics , Genes, T-Cell Receptor delta , Humans , Intraepithelial Lymphocytes/chemistry , Polymerase Chain Reaction/methods , Reproducibility of Results , Sequence Analysis, DNA/methodsABSTRACT
CCR9 expressed on T lymphocytes mediates migration to the small intestine in response to a gradient of CCL25. CCL25-stimulated activation of α4ß7 integrin promotes cell adherence to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expressed by vascular endothelial cells of the intestine, further mediating gut-specific homing. Inflammatory bowel disease is a chronic inflammatory condition that primarily affects the gastrointestinal tract and is characterized by leukocyte infiltration. Glucocorticoids (GCs) are widely used to treat inflammatory bowel disease but their effect on intestinal leukocyte homing is not well understood. We investigated the effect of GCs on the gut-specific chemokine receptor pair, CCR9 and CCL25. Using human peripheral blood-derived T lymphocytes enriched for CCR9 by cell sorting or culturing with all-trans retinoic acid, we measured chemotaxis, intracellular calcium flux, and α4ß7-mediated cell adhesion to plate-bound MAdCAM-1. Dexamethasone (DEX), a specific GC receptor agonist, significantly reduced CCR9-mediated chemotaxis and adhesion to MAdCAM-1 without affecting CCR9 surface expression. In contrast, in the same cells, DEX increased CXCR4 surface expression and CXCL12-mediated signaling and downstream functions. The effects of DEX on human primary T cells were reversed by the GC receptor antagonist mifepristone. These results demonstrate that GCs suppress CCR9-mediated chemotaxis, intracellular calcium flux, and α4ß7-mediated cell adhesion in vitro, and these effects could contribute to the efficacy of GCs in treating intestinal inflammation in vivo.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Dexamethasone/pharmacology , Immunoglobulins/metabolism , Inflammatory Bowel Diseases/drug therapy , Mucoproteins/metabolism , Receptors, Lymphocyte Homing/metabolism , T-Lymphocytes/drug effects , Calcium Signaling/drug effects , Cell Adhesion/drug effects , Cell Adhesion Molecules , Cell Movement/drug effects , Cells, Cultured , Chemokines, CC/metabolism , Chemotaxis/drug effects , Humans , Inflammatory Bowel Diseases/immunology , Receptors, CCR/metabolism , T-Lymphocytes/physiology , Tretinoin/metabolismABSTRACT
BACKGROUND: Patients with heterozygous germline mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) experience autoimmunity and lymphoid hyperplasia. OBJECTIVES: Because regulation of the phosphoinositide 3-kinase (PI3K) pathway is critical for maintaining regulatory T (Treg) cell functions, we investigate Treg cells in patients with heterozygous germline PTEN mutations (PTEN hamartoma tumor syndrome [PHTS]). METHODS: Patients with PHTS were assessed for immunologic conditions, lymphocyte subsets, forkhead box P3 (FOXP3)+ Treg cell levels, and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway, we assessed Treg cell induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunologic synapse. RESULTS: Autoimmunity and peripheral lymphoid hyperplasia were found in 43% of 79 patients with PHTS. Immune dysregulation in patients with PHTS included lymphopenia, CD4+ T-cell reduction, and changes in T- and B-cell subsets. Although total CD4+FOXP3+ Treg cell numbers are reduced, frequencies are maintained in the blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP) downstream of PTEN is highly expressed in normal human Treg cells and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Treg cells in vitro and Treg cell mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunologic synapse. CONCLUSION: Heterozygous loss of function of PTEN in human subjects has a significant effect on T- and B-cell immunity. Assembly of the PTEN-PHLPP phosphatase network allows coordinated phosphatase activities at the site of T-cell receptor activation, which is important for limiting PI3K hyperactivation in Treg cells despite PTEN haploinsufficiency.
Subject(s)
B-Lymphocytes/physiology , Hamartoma Syndrome, Multiple/immunology , Immunological Synapses/metabolism , Lymphocyte Subsets/physiology , Nuclear Proteins/metabolism , PTEN Phosphohydrolase/metabolism , Phosphoprotein Phosphatases/metabolism , T-Lymphocytes, Regulatory/physiology , Adolescent , Adult , Aged , Autoimmunity , Cells, Cultured , Child , Forkhead Transcription Factors/metabolism , Hamartoma Syndrome, Multiple/genetics , Humans , Hyperplasia , Male , Membrane Potential, Mitochondrial , Middle Aged , Mutation/genetics , PTEN Phosphohydrolase/genetics , Protein Binding , Protein Transport , Signal Transduction , Young AdultABSTRACT
Acute respiratory virus infections predispose the cystic fibrosis (CF) lung to chronic bacterial colonization, which contributes to high mortality. For reasons unknown, respiratory virus infections have a prolonged duration in CF. Here, we demonstrate that mice carrying the most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutation in humans, ΔF508, show increased morbidity and mortality following infection with a common human enterovirus. ΔF508 mice demonstrated impaired viral clearance, a slower type I interferon response and delayed production of virus-neutralizing antibodies. While the ΔF508 mice had a normal immune cell repertoire, unchanged serum immunoglobulin concentrations and an intact immune response to a T-cell-independent antigen, their response to a T-cell-dependent antigen was significantly delayed. Our studies reveal a novel function for CFTR in antiviral immunity and demonstrate that the ΔF508 mutation in cftr is coupled to an impaired adaptive immune response. This important insight could open up new approaches for patient care and treatment.
Subject(s)
Adaptive Immunity/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Cystic Fibrosis/immunology , Immunity, Innate/genetics , Mutation , Virus Diseases/etiology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Codon , Cystic Fibrosis/complications , Disease Models, Animal , Disease Resistance/genetics , Disease Resistance/immunology , Gene Expression Regulation , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immunization , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Interferon-alpha/biosynthesis , Mice , Poly I-C/immunology , Survival Rate , Viral LoadABSTRACT
HIV infection is associated with immune dysfunction, perturbation of immune-cell subsets and opportunistic infections. CD161++ CD8+ T cells are a tissue-infiltrating population that produce IL17A, IL22, IFN, and TNFα, cytokines important in mucosal immunity. In adults they dominantly express the semi-invariant TCR Vα7.2, the canonical feature of mucosal associated invariant T (MAIT) cells and have been recently implicated in host defense against pathogens. We analyzed the frequency and function of CD161++ /MAIT cells in peripheral blood and tissue from patients with early stage or chronic-stage HIV infection. We show that the CD161++ /MAIT cell population is significantly decreased in early HIV infection and fails to recover despite otherwise successful treatment. We provide evidence that CD161++ /MAIT cells are not preferentially infected but may be depleted through diverse mechanisms including accumulation in tissues and activation-induced cell death. This loss may impact mucosal defense and could be important in susceptibility to specific opportunistic infections in HIV.
Subject(s)
HIV Infections/immunology , Immunity, Mucosal/immunology , NK Cell Lectin-Like Receptor Subfamily B/immunology , T-Lymphocyte Subsets/immunology , Adult , Anti-HIV Agents/therapeutic use , Antiretroviral Therapy, Highly Active , Apoptosis/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/virology , Cells, Cultured , Cohort Studies , Escherichia coli/immunology , Female , Flow Cytometry , HIV/drug effects , HIV/immunology , HIV Infections/blood , HIV Infections/drug therapy , Humans , Immunohistochemistry , Interleukin-17/immunology , Interleukin-17/metabolism , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , Lymphocyte Count , Male , Middle Aged , NK Cell Lectin-Like Receptor Subfamily B/metabolism , Receptors, CCR5/immunology , Receptors, CCR5/metabolism , Receptors, CCR6/immunology , Receptors, CCR6/metabolism , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/virology , Time FactorsABSTRACT
CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A.
Subject(s)
Granzymes/administration & dosage , Leukemia, Myelomonocytic, Acute/drug therapy , Leukemia, Myelomonocytic, Chronic/drug therapy , Aged , Aged, 80 and over , Drug Stability , Female , HL-60 Cells , Humans , Immunotoxins/administration & dosage , Immunotoxins/blood , Immunotoxins/immunology , Immunotoxins/pharmacokinetics , Leukemia, Myelomonocytic, Acute/immunology , Leukemia, Myelomonocytic, Chronic/immunology , Male , Middle Aged , Receptors, IgG/biosynthesis , Receptors, IgG/immunology , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/blood , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacokinetics , Single-Chain Antibodies/administration & dosage , Single-Chain Antibodies/immunology , Single-Chain Antibodies/pharmacokineticsABSTRACT
Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (sALCL) are rare lymphoproliferative cancer types. Although most HL patients can be cured by chemo- and radio-therapy, 4-50% of patients relapse and have a poor prognosis. The need for improved therapeutic options for patients with relapsed or refractory disease has been addressed by CD30-specific antibody-based immunotherapeutics. However, available CD30-specific monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) or chimeric immunotoxins suffer from the requirement of a functional host immunity, undesirable immune reactions or heterogeneity and instability, respectively. Here, we present a new fusion protein comprised of the CD30-specific antibody single-chain fragment Ki4(scFv) and the human pro-apoptotic effector protein, microtubule-associated protein tau (MAPT). Ki4(scFv)-MAP selectively induced apoptosis in rapidly proliferating L540cy, L428, and Karpas 299 cells in a dose-dependent manner. Tubulin polymerization assays confirmed that Ki4(scFv)-MAP stabilizes microtubules, suggesting a mechanism for its pro-apoptotic action. Dose-finding experiments proved that Ki4(scFv)-MAP is well tolerated in mice compared to the previously reported Ki4(scFv)-ETA'. Ki4(scFv)-MAP significantly inhibited growth of subcutaneous L540cy xenograft tumours in mice. Our data present a novel approach for the treatment of CD30(+) lymphomas, combining the binding specificity of a target-specific antibody fragment with the selective cytotoxicity of MAPT towards proliferating lymphoma cells.
Subject(s)
Antibody-Dependent Cell Cytotoxicity , Hodgkin Disease/metabolism , Hodgkin Disease/pathology , Ki-1 Antigen/metabolism , Recombinant Fusion Proteins/pharmacology , Single-Chain Antibodies/pharmacology , tau Proteins/metabolism , Animals , Apoptosis/drug effects , Cell Line, Tumor , Disease Models, Animal , Female , Humans , Ki-1 Antigen/immunology , Mice , Microtubules/metabolism , Protein Binding , Protein Stability , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/immunology , Single-Chain Antibodies/metabolism , Tumor Burden/drug effects , Xenograft Model Antitumor Assays , tau Proteins/immunologyABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis and limited treatment options. Efforts to identify effective treatments are thwarted by limited understanding of IPF pathogenesis and poor translatability of available preclinical models. Here we generated spatially resolved transcriptome maps of human IPF (n = 4) and bleomycin-induced mouse pulmonary fibrosis (n = 6) to address these limitations. We uncovered distinct fibrotic niches in the IPF lung, characterized by aberrant alveolar epithelial cells in a microenvironment dominated by transforming growth factor beta signaling alongside predicted regulators, such as TP53 and APOE. We also identified a clear divergence between the arrested alveolar regeneration in the IPF fibrotic niches and the active tissue repair in the acutely fibrotic mouse lung. Our study offers in-depth insights into the IPF transcriptional landscape and proposes alveolar regeneration as a promising therapeutic strategy for IPF.
Subject(s)
Bleomycin , Idiopathic Pulmonary Fibrosis , Transcriptome , Animals , Humans , Mice , Idiopathic Pulmonary Fibrosis/genetics , Idiopathic Pulmonary Fibrosis/pathology , Bleomycin/toxicity , Disease Models, Animal , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/genetics , Lung/metabolism , Lung/pathology , Mice, Inbred C57BL , Signal Transduction/genetics , Male , Gene Expression Profiling , Alveolar Epithelial Cells/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Regeneration/genetics , Apolipoproteins E/geneticsABSTRACT
Rationale: Pulmonary surfactant is vital for lung homeostasis as it reduces surface tension to prevent alveolar collapse and provides essential immune-regulatory and antipathogenic functions. Previous studies demonstrated dysregulation of some individual surfactant components in COPD. We investigated relationships between COPD disease measures and dysregulation of surfactant components to gain new insights into potential disease mechanisms. Methods: Bronchoalveolar lavage proteome and lipidome were characterised in ex-smoking mild/moderate COPD subjects (n=26) and healthy ex-smoking (n=20) and never-smoking (n=16) controls using mass spectrometry. Serum surfactant protein analysis was performed. Results: Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, surfactant protein (SP)-B, SP-A and SP-D concentrations were lower in COPD versus controls (log2 fold change (log2FC) -2.0, -2.2, -1.5, -0.5, -0.7 and -0.5 (adjusted p<0.02), respectively) and correlated with lung function. Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D, napsin A and CD44 inversely correlated with computed tomography small airways disease measures (expiratory to inspiratory mean lung density) (r= -0.56, r= -0.58, r= -0.45, r= -0.36, r= -0.44, r= -0.37, r= -0.40 and r= -0.39 (adjusted p<0.05)). Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D and NAPSA inversely correlated with emphysema (% low-attenuation areas): r= -0.55, r= -0.61, r= -0.48, r= -0.51, r= -0.41, r= -0.31 and r= -0.34, respectively (adjusted p<0.05). Neutrophil elastase, known to degrade SP-A and SP-D, was elevated in COPD versus controls (log2FC 0.40, adjusted p=0.0390), and inversely correlated with SP-A and SP-D. Serum SP-D was increased in COPD versus healthy ex-smoking volunteers, and predicted COPD status (area under the curve 0.85). Conclusions: Using a multiomics approach, we demonstrate, for the first time, global surfactant dysregulation in COPD that was associated with emphysema, giving new insights into potential mechanisms underlying the cause or consequence of disease.
Subject(s)
Arthritis/immunology , Indazoles/pharmacology , Inflammatory Bowel Diseases/immunology , Nuclear Receptor Subfamily 1, Group F, Member 3/antagonists & inhibitors , Th17 Cells/drug effects , Th17 Cells/immunology , Arthritis/drug therapy , Arthritis/genetics , Arthritis/metabolism , Gene Expression Regulation/drug effects , Humans , Indazoles/chemistry , Inflammatory Bowel Diseases/drug therapy , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/metabolism , Interleukin-17/biosynthesis , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Th17 Cells/metabolismABSTRACT
In vivo optical Imaging is an inexpensive and highly sensitive modality to investigate and follow up diseases like breast cancer. However, fluorescence labels and specific tracers are still works in progress to bring this promising modality into the clinical day-to-day use. In this study an anti-MUC-1 binding single-chain antibody fragment was screened, produced and afterwards labeled with newly designed and surface modified NaYF(4):Yb,Er upconversion nanoparticles as fluorescence reporter constructs. The MUC-1 binding of the conjugate was examined in vitro and in vivo using modified state-of-the-art small animal Imaging equipment. Binding of the newly generated upconversion nanoparticle based probe to MUC-1 positive cells was clearly shown via laser scanning microscopy and in an initial proof of principal small animal optical imaging approach.
Subject(s)
Molecular Imaging/methods , Mucin-1/immunology , Nanoparticles , Optical Imaging/methods , Single-Chain Antibodies , Animals , Breast Neoplasms/diagnosis , Breast Neoplasms/immunology , Cell Line, Tumor , Female , Humans , MCF-7 Cells , Mice , Mice, Nude , Neoplasm Transplantation , Single-Chain Antibodies/immunology , Staining and Labeling , Transplantation, HeterologousABSTRACT
Target-specific acute myeloid leukemia (AML) immunotherapy requires selective cell-surface antigens on AML blast cells. CD64 is a promising candidate antigen because it is abundantly expressed on monocytoid differentiated AML subtypes. In previous studies, a chemically linked full-length anti-CD64 immunotoxin based on ricin A showed promising results in several animal models, but further development has been hindered by its substantial, dose-limiting off-target effects. We recently constructed the recombinant immunotoxin H22(scFv)-ETA', comprising a truncated Pseudomonas exotoxin A (PE) and a humanized scFv antibody against CD64. This molecule was shown to kill CD64(+) AML-derived tumor cell lines and primary patient-derived AML cells specifically, both in vitro and ex vivo. Here we describe the in vivo efficiency of H22(scFv)-ETA' in the U937/SCID mouse xenograft model for human AML, by providing immunohistochemical evidence for the elimination of human CD64(+) tumor cells in mouse organs. H22(scFv)-ETA' showed potent antitumor activity against myeloid tumor cells and significantly prolonged the overall survival of AML xenograft animals. In conclusion, H22(scFv)-ETA' is efficacious against AML with monocytoid differentiation in vitro and in animal models in vivo, providing the basis for a novel therapeutic strategy for the treatment of AML patients.
Subject(s)
ADP Ribose Transferases/immunology , Bacterial Toxins/immunology , Disease Models, Animal , Exotoxins/immunology , Immunotoxins/immunology , Leukemia, Myeloid, Acute/therapy , Recombinant Fusion Proteins/therapeutic use , Single-Chain Antibodies/immunology , Virulence Factors/immunology , ADP Ribose Transferases/genetics , Animals , Bacterial Toxins/genetics , Exotoxins/genetics , Female , Humans , Immunoenzyme Techniques , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Immunotoxins/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/immunology , Mice , Mice, Inbred NOD , Mice, SCID , Receptors, IgG/genetics , Receptors, IgG/immunology , Recombinant Fusion Proteins/immunology , Single-Chain Antibodies/genetics , Survival Rate , Tumor Cells, Cultured , Virulence Factors/genetics , Xenograft Model Antitumor Assays , Pseudomonas aeruginosa Exotoxin AABSTRACT
Coxsackieviruses (CV) are important human pathogens that have been implicated in the pathogenesis of several diseases, including myocarditis and pancreatitis. How the human immune system recognizes and controls CV infections is not well understood. Studies in mice suggest that natural killer (NK) cells play a critical role in viral clearance and host survival, but the mechanism(s) by which human NK cells may contribute to the host anti-CV defence has not been investigated. Here we show that CVB3 infection markedly reduces HLA class I cell surface expression but does not increase the expression of the activating NK cell receptor ligands MICA/B and ULBP1-3 on human cells. We also demonstrate that the lowered target cell HLA class I surface expression does not correlate with an increased susceptibility to NK cell-mediated killing. However, NK cells responded with a robust production of interferon gamma (IFN-gamma) when peripheral blood mononuclear cells were cocultured with infected cells. In summary, this study shows that CVB3 interferes with the expression of NK cell receptor ligands on infected cells and indicates that IFN-gamma production, rather than cytotoxicity, marks the early human NK cell response to CVB3 infection.
Subject(s)
Coxsackievirus Infections/immunology , Interferon-gamma/biosynthesis , Killer Cells, Natural/immunology , Capsid Proteins/analysis , Down-Regulation , HeLa Cells , Histocompatibility Antigens Class I/metabolism , Humans , Interferon-gamma/immunology , Killer Cells, Natural/virology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Ligands , Receptors, Immunologic/metabolismABSTRACT
Cystic fibrosis-related diabetes (CFRD) is a common complication for patients with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The cause of CFRD is unclear, but a commonly observed reduction in first-phase insulin secretion suggests defects at the beta cell level. Here we aimed to examine beta- and alpha-cell function in the Cftrtm1EUR/F508del mouse model (C57BL/6J), which carries the most common human mutation in CFTR, the F508del mutation. CFTR expression, beta cell mass, insulin granule distribution, hormone secretion and single cell capacitance changes were evaluated using islets (or beta cells) from F508del mice and age-matched wild-type mice aged 7-10 weeks. Granular pH was measured with DND-189 fluorescence. Serum glucose, insulin and glucagon levels were measured in vivo, and glucose tolerance was assessed using IPGTT. We show increased secretion of proinsulin and concomitant reduced secretion of C-peptide in islets from F508del mice compared to WT mice. Exocytosis and number of docked granules was reduced. We confirmed reduced granular pH by CFTR stimulation. We detected decreased pancreatic beta cell area, but unchanged beta cell number. Moreover, the F508del mutation caused failure to suppress glucagon secretion leading to hyperglucagonemia. In conclusion, F508del mice have beta cell defects resulting in 1) reduced number of docked insulin granules and reduced exocytosis, and 2) potential defective proinsulin cleavage and secretion of immature insulin. These observations provide insight into the functional role of CFTR in pancreatic islets and contribute to increased understanding of the pathogenesis of CFRD.
ABSTRACT
OBJECTIVES: The intestinal brush border enzymes alkaline sphingomyelinase (alk-SMase) and neutral ceramidase (CDase) digest milk sphingomyelin in suckling neonates. In addition, alk-SMase, CDase, and acid sphingomyelinase (acid-SMase) have been implicated in sphingolipid signaling, which exhibits abnormalities in cystic fibrosis (CF). In this study, we tested the hypothesis that the expression of these enzymes is different in CF. MATERIALS AND METHODS: We used mice with F508del (Cftr) mutation, a CF mouse model with well-characterized intestinal pathology. Enzyme activities were measured using radiolabeled sphingolipid substrates incubated with tissue homogenates from different organs and intestinal contents of wild-type mice, homozygous, and heterozygous F508del mice. RESULTS: No difference was found in levels of CDase and alk-SMase in the small intestinal mucosa or in their longitudinal distribution. Acid-SMase activity was significantly lower in the mucosa of the distal half of the small intestine of F508del compared with wild-type mice. Despite a lower body weight of F508del mice, length and weight of the small intestine and weight per centimeter of colon were larger than in wild-type. Neutral CDase and alk-SMase activities in lungs were lower than in the gut, whereas acid-SMase activity was comparable in both organs. CDase activity in the spleen was significantly higher in F508del than in wild-type mice. CONCLUSIONS: Alk-SMase and neutral CDase are normally expressed in F508del CF mice, whereas activity of acid-SMase in the distal small intestine is decreased. We found no differences in activity of these enzymes in lungs in this mouse model.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/enzymology , Cystic Fibrosis/genetics , Intestine, Small/enzymology , Lung/enzymology , Neutral Ceramidase/metabolism , Sphingomyelin Phosphodiesterase/metabolism , Animals , Body Weight , Colon/pathology , DNA Primers , Female , Humans , Intestine, Small/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microvilli/enzymology , Mutation , Organ Size , Sequence Deletion , Weight LossABSTRACT
CD64, the high affinity receptor for IgG (FcgammaRI) is expressed on acute myeloid leukemia blast cells and has recently been described as a specific target for immunotherapy. To generate a recombinant immunotoxin, the anti-CD64 single chain fragment (scFv) m22 was cloned into the bacterial expression vector pBM1.1 and fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Genetically modified Escherichia coli BL21 Star (DE3) were grown under osmotic stress conditions in the presence of compatible solutes. After isopropyl beta-D-thiogalactoside induction, the 70-kDa His(10)-tagged m22(scFv)-ETA' was directed into the periplasmic space and purified by a combination of metal-ion affinity and molecular size-chromatography. The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays, using CD64-positive AML cells. Binding specificity of m22(scFv)-ETA' was verified by competition with the parental anti-CD64 monoclonal antibody m22. The recombinant immunotoxin showed significant toxicity toward the CD64-positive cell lines HL-60 and U937 reaching 50% inhibition of cell proliferation at a concentration (IC(50)) of 11.6 ng/ml against HL-60 cells and 12.9 ng/ml against U937 cells. Approximately 41% of primary leukemia cells from a patient with CD64-positive AML were driven into early apoptosis by m22(scFv)-ETA' as measured by flow cytometric analysis. This is the first article documenting the specific cytotoxicity of a novel recombinant immunotoxin with major implications for immunotherapy of CD64-positive diseases.
Subject(s)
ADP Ribose Transferases/pharmacology , Bacterial Toxins/pharmacology , Exotoxins/pharmacology , Immunotoxins/pharmacology , Leukemia, Myeloid/drug therapy , Receptors, IgG/immunology , Virulence Factors/pharmacology , ADP Ribose Transferases/genetics , ADP Ribose Transferases/immunology , Adult , Antibody Specificity , Apoptosis/drug effects , Bacterial Toxins/genetics , Bacterial Toxins/immunology , Exotoxins/genetics , Exotoxins/immunology , Humans , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/immunology , Immunoglobulin Fragments/pharmacology , Immunotoxins/genetics , Immunotoxins/immunology , Leukemia, Myeloid/immunology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacology , Virulence Factors/genetics , Virulence Factors/immunology , Pseudomonas aeruginosa Exotoxin AABSTRACT
The IFIH1 gene encodes the pattern recognition receptor MDA5. A common polymorphism in IFIH1 (rs1990760, A946T) confers increased risk for autoimmune disease, including type 1-diabetes (T1D). Coxsackievirus infections are linked to T1D and cause beta-cell damage in vitro. Here we demonstrate that the rs1990760 polymorphism regulates the interferon (IFN) signature expressed by human pancreatic islets following Coxsackievirus infection. A strong IFN signature was associated with high expression of IFNλ1 and IFNλ2, linking rs1990760 to the expression of type III IFNs. In the high-responding genotype, IRF-1 expression correlated with that of type III IFN, suggesting a positive-feedback on type III IFN transcription. In summary, our study uncovers an influence of rs1990760 on the canonical effector function of MDA5 in response to an acute infection of primary human parenchymal cells with a clinically relevant virus linked to human T1D. It also highlights a previously unrecognized connection between the rs1990760 polymorphism and the expression level of type III IFNs.