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1.
Arthritis Rheumatol ; 70(10): 1597-1609, 2018 10.
Article in English | MEDLINE | ID: mdl-29687651

ABSTRACT

OBJECTIVE: Toll-like receptors (TLRs) 7 and 9 are important innate signaling molecules with opposing roles in the development and progression of systemic lupus erythematosus (SLE). While multiple studies support the notion of a dependency on TLR-7 for disease development, genetic ablation of TLR-9 results in severe disease with glomerulonephritis (GN) by a largely unknown mechanism. This study was undertaken to examine the suppressive role of TLR-9 in the development of severe lupus in a mouse model. METHODS: We crossed Sle1 lupus-prone mice with TLR-9-deficient mice to generate Sle1TLR-9-/- mice. Mice ages 4.5-6.5 months were evaluated for severe autoimmunity by assessing splenomegaly, GN, immune cell populations, autoantibody and total Ig profiles, kidney dendritic cell (DC) function, and TLR-7 protein expression. Mice ages 8-10 weeks were used for functional B cell studies, Ig profiling, and determination of TLR-7 expression. RESULTS: Sle1TLR-9-/- mice developed severe disease similar to TLR-9-deficient MRL and Nba2 models. Sle1TLR-9-/- mouse B cells produced more class-switched antibodies, and the autoantibody repertoire was skewed toward RNA-containing antigens. GN in these mice was associated with DC infiltration, and purified Sle1TLR-9-/- mouse renal DCs were more efficient at TLR-7-dependent antigen presentation and expressed higher levels of TLR-7 protein. Importantly, this increase in TLR-7 expression occurred prior to disease development, indicating a role in the initiation stages of tissue destruction. CONCLUSION: The increase in TLR-7-reactive immune complexes, and the concomitant enhanced expression of their receptor, promotes inflammation and disease in Sle1TLR9-/- mice.


Subject(s)
Lupus Erythematosus, Systemic/immunology , Lupus Nephritis/immunology , Toll-Like Receptor 7/immunology , Toll-Like Receptor 9/deficiency , Up-Regulation/immunology , Animals , Antigens/immunology , Disease Models, Animal , Mice , RNA/immunology , Toll-Like Receptor 9/immunology
2.
J Allergy Clin Immunol Pract ; 6(6): 1989-1998.e3, 2018.
Article in English | MEDLINE | ID: mdl-29627457

ABSTRACT

BACKGROUND: Patients with asthma and elevated blood eosinophils are at increased risk of severe exacerbations. Management of these patients should consider nonadherence to inhaled corticosteroid (ICS) therapy as a factor for increased exacerbation risk. OBJECTIVE: The objective of this study was to investigate whether poor adherence to ICS therapy explains the occurrence of asthma exacerbations in patients with elevated blood eosinophil levels. METHODS: This historical cohort study identified patients within the Optimum Patient Care Research Database, aged 18 years or more, at Global Initiative for Asthma step 3 or 4, with 2 or more ICS prescriptions during the year before the clinical review. Patient characteristics and adherence (based on prescription refills and patient self-report) for ICS therapy were analyzed for those with elevated (>400 cells/µL) or normal (≤400 cells/µL) blood eosinophils. RESULTS: We studied 7195 patients (66% female, mean age 60 years) with median eosinophil count of 200 cells/µL and found 81% to be not fully adherent to ICS therapy. A total of 1031 patients (14%) had elevated blood eosinophil counts (58% female, mean age 60 years), 83% of whom were not fully adherent to ICS. An increased proportion of adherent patients in the elevated blood eosinophil group had 2 or more exacerbations (14.0% vs 7.2%; P = .003) and uncontrolled asthma (73% vs 60.8%; P = .004) as compared with non-fully adherent patients. CONCLUSIONS: Approximately 1 in 7 patients had elevated eosinophils. Adherence to ICS therapy was not associated with decreased exacerbations for these patients. Additional therapy should be considered for these patients, such as biologics, which have been previously shown to improve control in severe uncontrolled eosinophilic asthma.


Subject(s)
Adrenal Cortex Hormones/therapeutic use , Asthma/drug therapy , Eosinophils/immunology , Medication Adherence/statistics & numerical data , Administration, Inhalation , Aged , Asthma/epidemiology , Cohort Studies , Disease Progression , Female , Humans , Male , Middle Aged , Risk , Treatment Outcome , United Kingdom/epidemiology
3.
Clin Rheumatol ; 37(3): 811-816, 2018 Mar.
Article in English | MEDLINE | ID: mdl-28593610

ABSTRACT

The signalling lymphocyte activation molecule (SLAM) family receptors play important roles in modulating immune responses. Previous studies in murine models and patients have suggested an association of the SLAM family (SLAMF) members with the development of autoimmunity, particularly systemic lupus erythematosus (SLE). Since previous investigations on CD244 expression have focussed on NK and T cells, the aim of this study was to evaluate the surface expression of major SLAMF members across monocytes and polymorphonuclear cells in an Asian SLE cohort and explore their potential associations with SLE-related disease activity and autoantibodies. Thirty-nine SLE patients and twenty-nine healthy controls (HC) were evaluated for the expression of CD150, CD84, CD229, CD48, CD244, CD352 and CD319. We determined a significantly lower expression of CD244 on monocytes in SLE patients compared to HC. Furthermore, monocyte CD244 expression was negatively associated with several serum autoantibody titres. Our findings suggest that this molecule plays an important role in immune tolerance mechanisms and should be investigated further.


Subject(s)
Blood Platelets/metabolism , Lupus Erythematosus, Systemic/metabolism , Monocytes/metabolism , Signaling Lymphocytic Activation Molecule Family/metabolism , Adult , Female , Humans , Male
4.
Rheumatology (Oxford) ; 56(6): 1025-1030, 2017 06 01.
Article in English | MEDLINE | ID: mdl-28137763

ABSTRACT

Objective: Siglecs are sialic acid-binding immunoglobulin-like lectins expressed on the surface of immune cells, which participate in the discrimination of self and non-self. We investigated myeloid CD33-related Siglec expression in a cohort of patients with SLE. Methods: Cell surface expression of Siglec-5/14, Siglec-9 and Siglec-10 on peripheral myeloid subsets were analysed from 39 SLE patients using flow cytometry. Genotyping of the Siglec-5/14 locus was also performed. Clinical markers of SLE disease activity, including SLEDAI, serum complement concentrations and serum autoantibodies, were assessed and correlated with Siglec levels. Results: Siglec-14 expression on SLE monocytes (median = 518, interquartile range: 411) was significantly higher when compared with healthy controls (median = 427, interquartile range: 289.3; P < 0.05) and correlated positively with SLEDAI scoring and anti-Sm and anti-SSB autoantibodies (P < 0.05). A negative correlation was determined with patient serum C3 concentrations (P < 0.005). Genotyping of the Siglec-5/14 locus revealed a high frequency of the Siglec-14 null allele across both groups, reflecting the incidence in Asian populations. Conclusion: Our data suggest that the Siglec immunomodulatory molecules, in particular Siglec-14 expression on monocytes, may play an important role in the inflammatory events of SLE. No bias was found with regard to SIGLEC14 genotype in our patient group compared with healthy controls. Larger comparisons of mixed ethnicity might, however, reveal an important role for Siglecs in the pathogenesis of autoimmune disease.


Subject(s)
Lectins/metabolism , Lupus Erythematosus, Systemic/metabolism , Receptors, Cell Surface/metabolism , Adult , Anti-Inflammatory Agents/therapeutic use , Case-Control Studies , Female , Genotype , Heterozygote , Humans , Lectins/genetics , Leukocytes, Mononuclear/metabolism , Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/ethnology , Male , Middle Aged , Polymorphism, Genetic , Prednisolone/therapeutic use , Receptors, Cell Surface/genetics , Up-Regulation/physiology , Young Adult
5.
Int Immunol ; 28(5): 223-32, 2016 05.
Article in English | MEDLINE | ID: mdl-26567289

ABSTRACT

The toll-like receptors (TLRs) are important innate receptors recognizing potentially pathogenic material. However, they also play a significant role in the development of Alzheimer's disease, cancer, autoimmunity and the susceptibility to viral infections. Macrophages are essential for an effective immune response to foreign material and the resolution of inflammation. In these studies, we examined the impact of different TLR ligands on macrophage cell function. We demonstrate that stimulation of all TLRs tested increases the phagocytosis of apoptotic cells by macrophages. TLR7 and TLR9 ligation decreased the levels of the surface co-expression molecules CD86 and MHCII, which was associated with a concomitant reduction in antigen presentation and proliferation of T cells. This down-regulation in macrophage function was not due to an increase in cell death. In fact, exposure to TLR7 or TLR9 ligands promoted cell viability for up to 9 days, in contrast to TLR3 or TLR4. Additionally, macrophages exposed to TLR7/TLR9 ligands had a significantly lower ratio of Il-12/Il-10 mRNA expression compared with those treated with the TLR4 ligand, LPS. Taken together, these data demonstrate that TLR7/TLR9 ligands push the macrophage into a phagocytic long-lived cell, with a decreased capacity of antigen presentation and reminiscent of the M2 polarized state.


Subject(s)
Antigen Presentation , Macrophages/immunology , Membrane Glycoproteins/agonists , Membrane Glycoproteins/immunology , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/immunology , Toll-Like Receptor 9/agonists , Toll-Like Receptor 9/immunology , Animals , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-12/genetics , Interleukin-12/immunology , Ligands , Lipopolysaccharides/toxicity , Membrane Glycoproteins/genetics , Mice , Mice, Knockout , Phagocytosis/drug effects , Phagocytosis/genetics , Toll-Like Receptor 7/genetics , Toll-Like Receptor 9/genetics
6.
Proc Natl Acad Sci U S A ; 112(45): E6195-204, 2015 Nov 10.
Article in English | MEDLINE | ID: mdl-26512111

ABSTRACT

Glomerulonephritis is a common and debilitating feature of systemic lupus erythematosus (SLE). The precise immune mechanisms that drive the progression from benign autoimmunity to glomerulonephritis are largely unknown. Previous investigations have shown that a moderate increase of the innate Toll-like receptor 7 (TLR7) is sufficient for the development of nephritis. In these systems normalization of B-cell TLR7 expression or temporal depletion of plasmacytoid dendritic cells (pDCs) slow progression; however, the critical cell that is responsible for driving full immunopathology remains unidentified. In this investigation we have shown that conventional DC expression of TLR7 is essential for severe autoimmunity in the Sle1Tg7 model of SLE. We show that a novel expanding CD11b(+) conventional DC subpopulation dominates the infiltrating renal inflammatory milieu, localizing to the glomeruli. Moreover, exposure of human myeloid DCs to IFN-α or Flu increases TLR7 expression, suggesting they may have a role in self-RNA recognition pathways in clinical disease. To our knowledge, this study is the first to highlight the importance of conventional DC-TLR7 expression for kidney pathogenesis in a murine model of SLE.


Subject(s)
Dendritic Cells/metabolism , Lupus Nephritis/physiopathology , Toll-Like Receptor 7/metabolism , Up-Regulation , Analysis of Variance , Animals , Base Sequence , CD11b Antigen/metabolism , DNA Primers/genetics , Flow Cytometry , Gene Expression Profiling/methods , Humans , Image Processing, Computer-Assisted , Kidney Glomerulus/cytology , Kidney Glomerulus/pathology , Lupus Nephritis/metabolism , Mice , Microscopy, Confocal , Molecular Sequence Data , Real-Time Polymerase Chain Reaction , Sequence Analysis, RNA , Statistics, Nonparametric
7.
J Biotechnol ; 146(4): 186-93, 2010 Apr 15.
Article in English | MEDLINE | ID: mdl-20156494

ABSTRACT

Gene amplification methodologies are frequently employed for the generation of large quantities of recombinant proteins in mammalian cells. Although they usually guarantee very high yields, they are very time consuming. In addition, due to the large genomic re-arrangements that frequently occur with amplification, the resulting high-producing clones can be unstable. We herein describe significant improvements to the dihydrofolate reductase (DHFR)/methotrexate (MTX) based gene amplification methodology typically employed to improve yields of recombinant proteins produced in genetically engineered CHO host cells. We demonstrate substantial synergy when such gene amplification is combined with extremely high codon optimisation strategies. As a result, expression saturation can be achieved rapidly, in as low as 5 nM MTX, with minimal effort and without compromise in final yields achieved.


Subject(s)
Codon , Gene Amplification , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Animals , CHO Cells , Cricetinae , Cricetulus , Gene Dosage , Methotrexate , Open Reading Frames , Recombinant Proteins/metabolism , Tetrahydrofolate Dehydrogenase
8.
J Clin Invest ; 118(9): 3143-50, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18688286

ABSTRACT

X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-beta region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.


Subject(s)
Chromosomes, Human, X , Genetic Therapy/adverse effects , Genetic Therapy/methods , Mutation , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/etiology , Severe Combined Immunodeficiency/therapy , Adaptor Proteins, Signal Transducing , Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinase Inhibitor p16/genetics , DNA-Binding Proteins/genetics , Follow-Up Studies , Humans , Infant , LIM Domain Proteins , Male , Metalloproteins/genetics , Models, Biological , Mutagenesis , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/complications , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Proto-Oncogene Proteins , Receptor, Notch1/genetics , Receptors, Interleukin-2/genetics , Severe Combined Immunodeficiency/complications
9.
Expert Opin Biol Ther ; 8(4): 397-407, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18352845

ABSTRACT

BACKGROUND: Primary immunodeficiencies (PID) are a group of inherited diseases that affect the development or activity of the immune system. In severe cases allogeneic haematopoietic stem cell transplantation has proved to be a successful curative modality but it is limited by toxicity and reduced efficacy in mismatched donor settings. OBJECTIVE: Gene therapy for PID has been developed as an alternative strategy and has entered the clinical arena. In this review we discuss the outcomes of recent gene therapy trials and some of the problems that remain to be tackled. METHODS: Results from clinical trials for X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficient SCID (ADA-SCID), and X-linked chronic granulomatous disease (X-CGD) are discussed. In addition, other conditions are highlighted such as the Wiskott Aldrich Syndrome (WAS) for which gene therapy has shown considerable promise in preclinical studies, and are currently being translated into novel clinical approaches. RESULTS/CONCLUSION: Whilst these encouraging results demonstrate that gene therapy can be used successfully to treat monogenic PID, the occurrence of vector-related side effects has highlighted the need for accurate assessment of the associated risks and a requirement for improvements in vector design.


Subject(s)
Genetic Therapy , Immunologic Deficiency Syndromes/therapy , Animals , Genetic Therapy/adverse effects , Genetic Therapy/methods , Genetic Vectors , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/therapy , Humans , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/immunology , Retroviridae/genetics , Severe Combined Immunodeficiency/genetics , Severe Combined Immunodeficiency/therapy , Treatment Outcome , Wiskott-Aldrich Syndrome/genetics , Wiskott-Aldrich Syndrome/therapy , X-Linked Combined Immunodeficiency Diseases/genetics , X-Linked Combined Immunodeficiency Diseases/therapy
10.
Mol Ther ; 16(3): 590-8, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18180772

ABSTRACT

Gene therapy for X-linked severe combined immunodeficiency (SCID-X1) has proven highly effective for long-term restoration of immunity in human subjects. However, the development of lymphoproliferative complications due to dysregulated proto-oncogene expression has underlined the necessity for developing safer vector systems. To reduce the potential for insertional mutagenesis, we have evaluated the efficacy of self-inactivating (SIN) gammaretroviral vectors in cellular and in vivo models of SCID-X1. Vectors incorporating an internal human elongation factor-1alpha regulatory element were capable of fully restoring the lymphoid differentiation potential of gammac-deficient lineage negative cells. Multilineage lymphoid reconstitution of a murine model was achieved at a similar level to that achieved by a conventional long-terminal repeat (LTR)-regulated vector used in previous clinical trials. Functional proliferative responses to mitogenic stimuli were also restored, and serum immunoglobulin levels were normalized. The reduced mutagenic potential conferred by SIN vector configurations and alternative non-LTR-based regulatory elements, together with proven efficacy in correction of cellular defects provides an important platform for development of the next phase of clinical trials for SCID-X1.


Subject(s)
Gammaretrovirus/genetics , Genetic Therapy/methods , Genetic Vectors/genetics , X-Linked Combined Immunodeficiency Diseases/therapy , Animals , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Blotting, Northern , Cell Differentiation , Cell Line , Cells, Cultured , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Humans , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Mice , Mice, Inbred C57BL , Models, Genetic , Polymerase Chain Reaction , Proto-Oncogene Mas , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , X-Linked Combined Immunodeficiency Diseases/genetics , X-Linked Combined Immunodeficiency Diseases/immunology
11.
Blood ; 110(5): 1448-57, 2007 Sep 01.
Article in English | MEDLINE | ID: mdl-17456723

ABSTRACT

Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy, we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters, the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters, suggesting a relative resistance to silencing. Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.


Subject(s)
Chromatin/genetics , Genetic Therapy , Genetic Vectors , Hematopoietic Stem Cells , Lentivirus , X-Linked Combined Immunodeficiency Diseases/therapy , Animals , Chromosomal Proteins, Non-Histone/genetics , Cytomegalovirus/genetics , Disease Models, Animal , Enhancer Elements, Genetic , Gene Expression , Gene Silencing , Genome, Viral/genetics , HeLa Cells , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Humans , Interleukin-2/genetics , K562 Cells , Lentivirus/genetics , Mice , Mice, SCID , Promoter Regions, Genetic/genetics , Signal Transduction/genetics , Spleen Focus-Forming Viruses/genetics , Transduction, Genetic , Transgenes/physiology , Virus Integration/genetics , X-Linked Combined Immunodeficiency Diseases/genetics
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