154 results on '"Chain BM"'
Search Results
2. Immunomodulation using bacterial enterotoxins
- Author
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Simmons, CP, Ghaem-Magami, M, Petrovska, L, Lopes, L, Chain, BM, Williams, NA, and Dougan, G
- Subjects
bacteria - Abstract
Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination by a mucosal route can effectively induce immune suppression. However, some bacterial-derived proteins, e.g. cholera toxin and the heat labile toxin of Escherichia coli, are immunogenic and immunomodulatory at mucosal surfaces and can effectively adjuvant immune responses to codelivered bystander antigens. This review summarizes some of the structural and biological characteristics of these toxins and provides examples of how these properties have been exploited for tolerance induction and mucosal vaccine development.
- Published
- 2016
3. S36 Differentiation of tuberculosis and sarcoidosis by transcriptional profiling of immune responses in mediastinal lymph node samples
- Author
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Tomlinson, GS, primary, Hardavella, G, additional, Brown, J, additional, Succony, L, additional, Navani, N, additional, Thomas, N, additional, Chain, BM, additional, Janes, SM, additional, and Noursadeghi, M, additional
- Published
- 2013
- Full Text
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4. CHEMICALLY MODIFIED ANTIGEN-PRESENTING CELLS INDUCE LYMPHOCYTE-T ALLOSPECIFIC HYPORESPONSIVENESS
- Author
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Mohammad Ibrahim, Coode, Wk, Takei, F., Ellis, Js, Chain, Bm, and Katz, Dr
5. Mucosal and systemic immune correlates of viral control after SARS-CoV-2 infection challenge in seronegative adults.
- Author
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Wagstaffe HR, Thwaites RS, Reynaldi A, Sidhu JK, McKendry R, Ascough S, Papargyris L, Collins AM, Xu J, Lemm NM, Siggins MK, Chain BM, Killingley B, Kalinova M, Mann A, Catchpole A, Davenport MP, Openshaw PJM, and Chiu C
- Subjects
- Humans, SARS-CoV-2, Vaccination, CD8-Positive T-Lymphocytes, Nasal Mucosa, COVID-19
- Abstract
Human infection challenge permits in-depth, early, and pre-symptomatic characterization of the immune response, enabling the identification of factors that are important for viral clearance. Here, we performed intranasal inoculation of 34 young adult, seronegative volunteers with a pre-Alpha severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain. Of these participants, 18 (53%) became infected and showed an interferon-dominated mediator response with divergent kinetics between nasal and systemic sites. Peripheral CD4
+ and CD8+ T cell activation and proliferation were early and robust but showed distinct kinetic and phenotypic profiles; antigen-specific T cells were largely CD38+ Ki67+ and displayed central and effector memory phenotypes. Both mucosal and systemic antibodies became detectable around day 10, but nasal antibodies plateaued after day 14 while circulating antibodies continued to rise. Intensively granular measurements in nasal mucosa and blood allowed modeling of immune responses to primary SARS-CoV-2 infection that revealed CD8+ T cell responses and early mucosal IgA responses strongly associated with viral control, indicating that these mechanisms should be targeted for transmission-reducing intervention.- Published
- 2024
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6. Single-cell analysis of bronchoalveolar cells in inflammatory and fibrotic post-COVID lung disease.
- Author
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Mehta P, Sanz-Magallón Duque de Estrada B, Denneny EK, Foster K, Turner CT, Mayer A, Milighetti M, Platé M, Worlock KB, Yoshida M, Brown JS, Nikolić MZ, Chain BM, Noursadeghi M, Chambers RC, Porter JC, and Tomlinson GS
- Subjects
- Humans, Male, Female, Middle Aged, Receptors, Antigen, T-Cell immunology, Receptors, Antigen, T-Cell metabolism, Receptors, Antigen, T-Cell genetics, Pulmonary Fibrosis immunology, Pulmonary Fibrosis etiology, Pulmonary Fibrosis pathology, CD8-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes immunology, Lung immunology, Lung pathology, Lung diagnostic imaging, Aged, Adult, Inflammation immunology, Inflammation pathology, Bronchoalveolar Lavage Fluid immunology, Bronchoalveolar Lavage Fluid cytology, Memory T Cells immunology, Transcriptome, COVID-19 immunology, COVID-19 pathology, Single-Cell Analysis, SARS-CoV-2 immunology
- Abstract
Background: Persistent radiological lung abnormalities are evident in many survivors of acute coronavirus disease 2019 (COVID-19). Consolidation and ground glass opacities are interpreted to indicate subacute inflammation whereas reticulation is thought to reflect fibrosis. We sought to identify differences at molecular and cellular level, in the local immunopathology of post-COVID inflammation and fibrosis., Methods: We compared single-cell transcriptomic profiles and T cell receptor (TCR) repertoires of bronchoalveolar cells obtained from convalescent individuals with each radiological pattern, targeting lung segments affected by the predominant abnormality., Results: CD4 central memory T cells and CD8 effector memory T cells were significantly more abundant in those with inflammatory radiology. Clustering of similar TCRs from multiple donors was a striking feature of both phenotypes, consistent with tissue localised antigen-specific immune responses. There was no enrichment for known SARS-CoV-2-reactive TCRs, raising the possibility of T cell-mediated immunopathology driven by failure in immune self-tolerance., Conclusions: Post-COVID radiological inflammation and fibrosis show evidence of shared antigen-specific T cell responses, suggesting a role for therapies targeting T cells in limiting post-COVID lung damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mehta, Sanz-Magallón Duque de Estrada, Denneny, Foster, Turner, Mayer, Milighetti, Platé, Worlock, Yoshida, Brown, Nikolić, Chain, Noursadeghi, Chambers, Porter and Tomlinson.)
- Published
- 2024
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7. Tissue CD14 + CD8 + T cells reprogrammed by myeloid cells and modulated by LPS.
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Pallett LJ, Swadling L, Diniz M, Maini AA, Schwabenland M, Gasull AD, Davies J, Kucykowicz S, Skelton JK, Thomas N, Schmidt NM, Amin OE, Gill US, Stegmann KA, Burton AR, Stephenson E, Reynolds G, Whelan M, Sanchez J, de Maeyer R, Thakker C, Suveizdyte K, Uddin I, Ortega-Prieto AM, Grant C, Froghi F, Fusai G, Lens S, Pérez-Del-Pulgar S, Al-Akkad W, Mazza G, Noursadeghi M, Akbar A, Kennedy PTF, Davidson BR, Prinz M, Chain BM, Haniffa M, Gilroy DW, Dorner M, Bengsch B, Schurich A, and Maini MK
- Subjects
- Humans, Neoplasms immunology, Neoplasms pathology, Receptors, Antigen, T-Cell immunology, Receptors, Antigen, T-Cell metabolism, Interleukin-2 biosynthesis, Interleukin-2 immunology, Chemotaxis, Leukocyte, Bacteria immunology, Intestines immunology, Intestines microbiology, CD8-Positive T-Lymphocytes cytology, CD8-Positive T-Lymphocytes drug effects, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes metabolism, Lipopolysaccharide Receptors metabolism, Lipopolysaccharides immunology, Lipopolysaccharides pharmacology, Myeloid Cells immunology, Myeloid Cells metabolism, Immune Tolerance drug effects, Immune Tolerance immunology, Liver drug effects, Liver immunology, Liver pathology, Liver virology
- Abstract
The liver is bathed in bacterial products, including lipopolysaccharide transported from the intestinal portal vasculature, but maintains a state of tolerance that is exploited by persistent pathogens and tumours
1-4 . The cellular basis mediating this tolerance, yet allowing a switch to immunity or immunopathology, needs to be better understood for successful immunotherapy of liver diseases. Here we show that a variable proportion of CD8+ T cells compartmentalized in the human liver co-stain for CD14 and other prototypic myeloid membrane proteins and are enriched in close proximity to CD14high myeloid cells in hepatic zone 2. CD14+ CD8+ T cells preferentially accumulate within the donor pool in liver allografts, among hepatic virus-specific and tumour-infiltrating responses, and in cirrhotic ascites. CD14+ CD8+ T cells exhibit increased turnover, activation and constitutive immunomodulatory features with high homeostatic IL-10 and IL-2 production ex vivo, and enhanced antiviral/anti-tumour effector function after TCR engagement. This CD14+ CD8+ T cell profile can be recapitulated by the acquisition of membrane proteins-including the lipopolysaccharide receptor complex-from mononuclear phagocytes, resulting in augmented tumour killing by TCR-redirected T cells in vitro. CD14+ CD8+ T cells express integrins and chemokine receptors that favour interactions with the local stroma, which can promote their induction through CXCL12. Lipopolysaccharide can also increase the frequency of CD14+ CD8+ T cells in vitro and in vivo, and skew their function towards the production of chemotactic and regenerative cytokines. Thus, bacterial products in the gut-liver axis and tissue stromal factors can tune liver immunity by driving myeloid instruction of CD8+ T cells with immunomodulatory ability., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2023
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8. Climate Change Affects Health: Are We Listening?
- Author
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Chain GS, Chain BM, and Pelliccia FB
- Abstract
Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
- Published
- 2022
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9. Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections.
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Chandran A, Rosenheim J, Nageswaran G, Swadling L, Pollara G, Gupta RK, Burton AR, Guerra-Assunção JA, Woolston A, Ronel T, Pade C, Gibbons JM, Sanz-Magallon Duque De Estrada B, Robert de Massy M, Whelan M, Semper A, Brooks T, Altmann DM, Boyton RJ, McKnight Á, Captur G, Manisty C, Treibel TA, Moon JC, Tomlinson GS, Maini MK, Chain BM, and Noursadeghi M
- Subjects
- CD8-Positive T-Lymphocytes, Flow Cytometry, Humans, SARS-CoV-2 genetics, COVID-19, Interferon Type I
- Abstract
Effective control of SARS-CoV-2 infection on primary exposure may reveal correlates of protective immunity to future variants, but we lack insights into immune responses before or at the time virus is first detected. We use blood transcriptomics, multiparameter flow cytometry, and T cell receptor (TCR) sequencing spanning the time of incident non-severe infection in unvaccinated virus-naive individuals to identify rapid type 1 interferon (IFN) responses common to other acute respiratory viruses and cell proliferation responses that discriminate SARS-CoV-2 from other viruses. These peak by the time the virus is first detected and sometimes precede virus detection. Cell proliferation is most evident in CD8 T cells and associated with specific expansion of SARS-CoV-2-reactive TCRs, in contrast to virus-specific antibodies, which lag by 1-2 weeks. Our data support a protective role for early type 1 IFN and CD8 T cell responses, with implications for development of universal T cell vaccines., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)
- Published
- 2022
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10. Application of T cell receptor (TCR) repertoire analysis for the advancement of cancer immunotherapy.
- Author
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Joshi K, Milighetti M, and Chain BM
- Subjects
- Humans, Immunotherapy, Adoptive, Receptors, Antigen, T-Cell genetics, T-Lymphocytes, Immunotherapy, Neoplasms therapy
- Abstract
T cell receptor (TCR) sequencing has emerged as a powerful new technology in analysis of the host-tumour interaction. The advances in NextGen sequencing technologies, coupled with powerful novel bioinformatic tools, allow quantitative and reproducible characterisation of repertoires from tumour and blood samples from an increasing number of patients with a variety of solid cancers. In this review, we consider how global metrics such as T cell clonality and diversity can be extracted from these repertoires and used to give insight into the mechanism of action of immune checkpoint blockade. Furthermore, we explore how the analysis of TCR overlap between repertories can help define spatial and temporal heterogeneity of the anti-tumoural immune response. Finally, we review how analysis of TCR sequence and structure, either of individual TCRs or from sets of related TCRs can be used to annotate the antigenic specificity, with important implications for the development of personalised adoptive cellular immunotherapies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2022
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11. Pre-existing polymerase-specific T cells expand in abortive seronegative SARS-CoV-2.
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Swadling L, Diniz MO, Schmidt NM, Amin OE, Chandran A, Shaw E, Pade C, Gibbons JM, Le Bert N, Tan AT, Jeffery-Smith A, Tan CCS, Tham CYL, Kucykowicz S, Aidoo-Micah G, Rosenheim J, Davies J, Johnson M, Jensen MP, Joy G, McCoy LE, Valdes AM, Chain BM, Goldblatt D, Altmann DM, Boyton RJ, Manisty C, Treibel TA, Moon JC, van Dorp L, Balloux F, McKnight Á, Noursadeghi M, Bertoletti A, and Maini MK
- Subjects
- Cell Proliferation, Cohort Studies, DNA-Directed RNA Polymerases metabolism, Evolution, Molecular, Female, Health Personnel, Humans, Male, Membrane Proteins immunology, Memory T Cells cytology, Multienzyme Complexes immunology, SARS-CoV-2 enzymology, SARS-CoV-2 growth & development, Transcription, Genetic immunology, Asymptomatic Infections, COVID-19 immunology, COVID-19 virology, DNA-Directed RNA Polymerases immunology, Memory T Cells immunology, SARS-CoV-2 immunology, Seroconversion
- Abstract
Individuals with potential exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) do not necessarily develop PCR or antibody positivity, suggesting that some individuals may clear subclinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections
1-3 . Here we hypothesize that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-2 (refs.4-11 ), would expand in vivo to support rapid viral control, aborting infection. We measured SARS-CoV-2-reactive T cells, including those against the early transcribed replication-transcription complex (RTC)12,13 , in intensively monitored healthcare workers (HCWs) who tested repeatedly negative according to PCR, antibody binding and neutralization assays (seronegative HCWs (SN-HCWs)). SN-HCWs had stronger, more multispecific memory T cells compared with a cohort of unexposed individuals from before the pandemic (prepandemic cohort), and these cells were more frequently directed against the RTC than the structural-protein-dominated responses observed after detectable infection (matched concurrent cohort). SN-HCWs with the strongest RTC-specific T cells had an increase in IFI27, a robust early innate signature of SARS-CoV-2 (ref.14 ), suggesting abortive infection. RNA polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and SARS-CoV-2 clades. RNA polymerase was preferentially targeted (among the regions tested) by T cells from prepandemic cohorts and SN-HCWs. RTC-epitope-specific T cells that cross-recognized HCoV variants were identified in SN-HCWs. Enriched pre-existing RNA-polymerase-specific T cells expanded in vivo to preferentially accumulate in the memory response after putative abortive compared to overt SARS-CoV-2 infection. Our data highlight RTC-specific T cells as targets for vaccines against endemic and emerging Coronaviridae., (© 2021. The Author(s).)- Published
- 2022
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12. Blood transcriptional biomarkers of acute viral infection for detection of pre-symptomatic SARS-CoV-2 infection: a nested, case-control diagnostic accuracy study.
- Author
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Gupta RK, Rosenheim J, Bell LC, Chandran A, Guerra-Assuncao JA, Pollara G, Whelan M, Artico J, Joy G, Kurdi H, Altmann DM, Boyton RJ, Maini MK, McKnight A, Lambourne J, Cutino-Moguel T, Manisty C, Treibel TA, Moon JC, Chain BM, and Noursadeghi M
- Subjects
- Adolescent, Adult, Biomarkers, Case-Control Studies, Female, Humans, Male, Middle Aged, Prospective Studies, SARS-CoV-2 genetics, Sensitivity and Specificity, COVID-19 diagnosis
- Abstract
Background: We hypothesised that host-response biomarkers of viral infections might contribute to early identification of individuals infected with SARS-CoV-2, which is critical to breaking the chains of transmission. We aimed to evaluate the diagnostic accuracy of existing candidate whole-blood transcriptomic signatures for viral infection to predict positivity of nasopharyngeal SARS-CoV-2 PCR testing., Methods: We did a nested case-control diagnostic accuracy study among a prospective cohort of health-care workers (aged ≥18 years) at St Bartholomew's Hospital (London, UK) undergoing weekly blood and nasopharyngeal swab sampling for whole-blood RNA sequencing and SARS-CoV-2 PCR testing, when fit to attend work. We identified candidate blood transcriptomic signatures for viral infection through a systematic literature search. We searched MEDLINE for articles published between database inception and Oct 12, 2020, using comprehensive MeSH and keyword terms for "viral infection", "transcriptome", "biomarker", and "blood". We reconstructed signature scores in blood RNA sequencing data and evaluated their diagnostic accuracy for contemporaneous SARS-CoV-2 infection, compared with the gold standard of SARS-CoV-2 PCR testing, by quantifying the area under the receiver operating characteristic curve (AUROC), sensitivities, and specificities at a standardised Z score of at least 2 based on the distribution of signature scores in test-negative controls. We used pairwise DeLong tests compared with the most discriminating signature to identify the subset of best performing biomarkers. We evaluated associations between signature expression, viral load (using PCR cycle thresholds), and symptom status visually and using Spearman rank correlation. The primary outcome was the AUROC for discriminating between samples from participants who tested negative throughout the study (test-negative controls) and samples from participants with PCR-confirmed SARS-CoV-2 infection (test-positive participants) during their first week of PCR positivity., Findings: We identified 20 candidate blood transcriptomic signatures of viral infection from 18 studies and evaluated their accuracy among 169 blood RNA samples from 96 participants over 24 weeks. Participants were recruited between March 23 and March 31, 2020. 114 samples were from 41 participants with SARS-CoV-2 infection, and 55 samples were from 55 test-negative controls. The median age of participants was 36 years (IQR 27-47) and 69 (72%) of 96 were women. Signatures had little overlap of component genes, but were mostly correlated as components of type I interferon responses. A single blood transcript for IFI27 provided the highest accuracy for discriminating between test-negative controls and test-positive individuals at the time of their first positive SARS-CoV-2 PCR result, with AUROC of 0·95 (95% CI 0·91-0·99), sensitivity 0·84 (0·70-0·93), and specificity 0·95 (0·85-0·98) at a predefined threshold (Z score >2). The transcript performed equally well in individuals with and without symptoms. Three other candidate signatures (including two to 48 transcripts) had statistically equivalent discrimination to IFI27 (AUROCs 0·91-0·95)., Interpretation: Our findings support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection for screening individuals at high risk of infection, such as contacts of index cases, to facilitate early case isolation and early use of antiviral treatments as they emerge., Funding: Barts Charity, Wellcome Trust, and National Institute of Health Research., Competing Interests: We declare no competing interests., (© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.)
- Published
- 2021
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13. Exaggerated IL-17A activity in human in vivo recall responses discriminates active tuberculosis from latent infection and cured disease.
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Pollara G, Turner CT, Rosenheim J, Chandran A, Bell LCK, Khan A, Patel A, Peralta LF, Folino A, Akarca A, Venturini C, Baker T, Ecker S, Ricciardolo FLM, Marafioti T, Ugarte-Gil C, Moore DAJ, Chain BM, Tomlinson GS, and Noursadeghi M
- Subjects
- Humans, Mycobacterium tuberculosis, Interleukin-17 immunology, Latent Tuberculosis diagnosis, Latent Tuberculosis immunology, Tuberculosis drug therapy, Tuberculosis immunology
- Abstract
Host immune responses at the site of Mycobacterium tuberculosis infection can mediate pathogenesis of tuberculosis (TB) and onward transmission of infection. We hypothesized that pathological immune responses would be enriched at the site of host-pathogen interactions modeled by a standardized tuberculin skin test (TST) challenge in patients with active TB compared to those without disease, and interrogated immune responses by genome-wide transcriptional profiling. We show exaggerated interleukin-17A (IL-17A) and T helper 17 (T
H 17) responses among 48 individuals with active TB compared to 191 with latent TB infection, associated with increased neutrophil recruitment and matrix metalloproteinase-1 expression, both involved in TB pathogenesis. Curative antimicrobial treatment reversed these observed changes. Increased IL-1β and IL-6 responses to mycobacterial stimulation were evident both in circulating monocytes and in molecular changes at the site of TST in individuals with active TB, supporting a model in which monocyte-derived IL-1β and IL-6 promote TH 17 differentiation within tissues. Modulation of these cytokine pathways may provide a rational strategy for host-directed therapy in active TB., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)- Published
- 2021
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14. Blood Transcriptomic Stratification of Short-term Risk in Contacts of Tuberculosis.
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Roe J, Venturini C, Gupta RK, Gurry C, Chain BM, Sun Y, Southern J, Jackson C, Lipman MC, Miller RF, Martineau AR, Abubakar I, and Noursadeghi M
- Subjects
- Case-Control Studies, Humans, Interferon-gamma Release Tests, Prospective Studies, Transcriptome, Mycobacterium tuberculosis genetics, Tuberculosis diagnosis, Tuberculosis epidemiology
- Abstract
Background: The highest risk of tuberculosis arises in the first few months after exposure. We reasoned that this risk reflects incipient disease among tuberculosis contacts. Blood transcriptional biomarkers of tuberculosis may predate clinical diagnosis, suggesting they offer improved sensitivity to detect subclinical incipient disease. Therefore, we sought to test the hypothesis that refined blood transcriptional biomarkers of active tuberculosis will improve stratification of short-term disease risk in tuberculosis contacts., Methods: We combined analysis of previously published blood transcriptomic data with new data from a prospective human immunodeficiency virus (HIV)-negative UK cohort of 333 tuberculosis contacts. We used stability selection as an alternative computational approach to identify an optimal signature for short-term risk of active tuberculosis and evaluated its predictive value in independent cohorts., Results: In a previously published HIV-negative South African case-control study of patients with asymptomatic Mycobacterium tuberculosis infection, a novel 3-gene transcriptional signature comprising BATF2, GBP5, and SCARF1 achieved a positive predictive value (PPV) of 23% for progression to active tuberculosis within 90 days. In a new UK cohort of 333 HIV-negative tuberculosis contacts with a median follow-up of 346 days, this signature achieved a PPV of 50% (95% confidence interval [CI], 15.7-84.3) and negative predictive value of 99.3% (95% CI, 97.5-99.9). By comparison, peripheral blood interferon gamma release assays in the same cohort achieved a PPV of 5.6% (95% CI, 2.1-11.8)., Conclusions: This blood transcriptional signature provides unprecedented opportunities to target therapy among tuberculosis contacts with greatest risk of incident disease., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)
- Published
- 2020
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15. Framework engineering to produce dominant T cell receptors with enhanced antigen-specific function.
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Thomas S, Mohammed F, Reijmers RM, Woolston A, Stauss T, Kennedy A, Stirling D, Holler A, Green L, Jones D, Matthews KK, Price DA, Chain BM, Heemskerk MHM, Morris EC, Willcox BE, and Stauss HJ
- Subjects
- Animals, Antigens, CD metabolism, Antigens, Differentiation, T-Lymphocyte metabolism, Cell Line, Tumor, Cell Proliferation, Cytokines metabolism, Gene Expression, Genetic Therapy, Humans, Lectins, C-Type metabolism, Lymphocyte Activation, Male, Mice, Mice, Inbred NOD, Mice, SCID, Models, Molecular, Protein Domains, Protein Engineering, Receptors, Antigen, T-Cell chemistry, Receptors, Antigen, T-Cell immunology, T-Lymphocytes immunology, Antigens immunology, Cell Engineering, Genes, T-Cell Receptor genetics, Genes, T-Cell Receptor immunology, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell metabolism, T-Lymphocytes metabolism
- Abstract
TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans.
- Published
- 2019
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16. Urine-derived lymphocytes as a non-invasive measure of the bladder tumor immune microenvironment.
- Author
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Wong YNS, Joshi K, Khetrapal P, Ismail M, Reading JL, Sunderland MW, Georgiou A, Furness AJS, Ben Aissa A, Ghorani E, Oakes T, Uddin I, Tan WS, Feber A, McGovern U, Swanton C, Freeman A, Marafioti T, Briggs TP, Kelly JD, Powles T, Peggs KS, Chain BM, Linch MD, and Quezada SA
- Subjects
- CD4-Positive T-Lymphocytes metabolism, CD4-Positive T-Lymphocytes pathology, CD8-Positive T-Lymphocytes metabolism, CD8-Positive T-Lymphocytes pathology, Female, Humans, Lymphocyte Count, Male, Urinary Bladder Neoplasms pathology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Tumor Microenvironment immunology, Urinary Bladder Neoplasms immunology, Urinary Bladder Neoplasms urine, Urine
- Abstract
Despite the advances in cancer immunotherapy, only a fraction of patients with bladder cancer exhibit responses to checkpoint blockade, highlighting a need to better understand drug resistance and identify rational immunotherapy combinations. However, accessibility to the tumor prior and during therapy is a major limitation in understanding the immune tumor microenvironment (TME). Herein, we identified urine-derived lymphocytes (UDLs) as a readily accessible source of T cells in 32 patients with muscle invasive bladder cancer (MIBC). We observed that effector CD8
+ and CD4+ cells and regulatory T cells within the urine accurately map the immune checkpoint landscape and T cell receptor repertoire of the TME. Finally, an increased UDL count, specifically high expression of PD-1 (PD-1hi ) on CD8+ at the time of cystectomy, was associated with a shorter recurrence-free survival. UDL analysis represents a dynamic liquid biopsy that is representative of the bladder immune TME that may be used to identify actionable immuno-oncology (IO) targets with potential prognostic value in MIBC., (© 2018 Wong et al.)- Published
- 2018
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17. The "Achilles' Heel" of Cancer and Its Implications for the Development of Novel Immunotherapeutic Strategies.
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Joshi K, Chain BM, Peggs KS, and Quezada SA
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- Animals, Antigens, Neoplasm immunology, Antineoplastic Agents, Immunological pharmacology, Humans, Immunity, Humoral, Immunologic Surveillance, Immunotherapy methods, Mutation immunology, Antineoplastic Agents, Immunological therapeutic use, Neoplasms immunology, Neoplasms therapy, Tumor Microenvironment
- Abstract
Over the last century, scientists have embraced the idea of mobilizing antitumor immune responses in patients with cancer. In the last decade, we have seen the rebirth of cancer immunotherapy and its validation in a series of high profile clinical trials following the discovery of several immune-regulatory receptors. Recent studies point toward the tumor mutational load and resulting neoantigen burden as being crucial to tumor cell recognition by the immune system, highlighting a potentially targetable Achilles' heel in cancer. In this review, we explore the key mechanisms that underpin the recognition of cancerous cells by the immune system and discuss how we may advance immunotherapeutic strategies to target the cancer mutanome to stimulate tumor-specific immune responses, ultimately, to improve the clinical outcome for patients with cancer., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)
- Published
- 2018
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18. Validation of Immune Cell Modules in Multicellular Transcriptomic Data.
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Pollara G, Murray MJ, Heather JM, Byng-Maddick R, Guppy N, Ellis M, Turner CT, Chain BM, and Noursadeghi M
- Subjects
- Cell Count, Gene Expression Profiling, Gene Expression Regulation, Humans, Lymph Nodes metabolism, Reproducibility of Results, Skin metabolism, Leukocytes metabolism, Transcriptome immunology
- Abstract
Numerous gene signatures, or modules have been described to evaluate the immune cell composition in transcriptomes of multicellular tissue samples. However, significant diversity in module gene content for specific cell types is associated with heterogeneity in their performance. In order to rank modules that best reflect their purported association, we have generated the modular discrimination index (MDI) score that assesses expression of each module in the target cell type relative to other cells. We demonstrate that MDI scores predict modules that best reflect independently validated differences in cellular composition, and correlate with the covariance between cell numbers and module expression in human blood and tissue samples. Our analyses demonstrate that MDI scores provide an ordinal summary statistic that reliably ranks the accuracy of gene expression modules for deconvolution of cell type abundance in transcriptional data., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2017
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19. In Vivo Molecular Dissection of the Effects of HIV-1 in Active Tuberculosis.
- Author
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Bell LC, Pollara G, Pascoe M, Tomlinson GS, Lehloenya RJ, Roe J, Meldau R, Miller RF, Ramsay A, Chain BM, Dheda K, and Noursadeghi M
- Subjects
- Adult, Aged, CD4 Lymphocyte Count, CD4-Positive T-Lymphocytes immunology, Computational Biology, Female, Gene Expression Profiling, HIV Infections complications, HIV Infections pathology, HIV Seropositivity, Humans, Immune Reconstitution Inflammatory Syndrome immunology, Interferon Type I immunology, Interleukin-10 immunology, Male, Middle Aged, Mycobacterium tuberculosis genetics, Tuberculosis complications, Tuberculosis pathology, Tuberculosis virology, Tuberculosis, Pulmonary complications, Tuberculosis, Pulmonary pathology, Tuberculosis, Pulmonary virology, Young Adult, HIV Infections immunology, HIV-1 immunology, Mycobacterium tuberculosis immunology, Tuberculosis immunology, Tuberculosis, Pulmonary immunology
- Abstract
Increased risk of tuberculosis (TB) associated with HIV-1 infection is primarily attributed to deficient T helper (Th)1 immune responses, but most people with active TB have robust Th1 responses, indicating that these are not sufficient to protect against disease. Recent findings suggest that favourable outcomes following Mycobacterium tuberculosis infection arise from finely balanced inflammatory and regulatory pathways, achieving pathogen control without immunopathology. We hypothesised that HIV-1 and antiretroviral therapy (ART) exert widespread changes to cell mediated immunity, which may compromise the optimal host protective response to TB and provide novel insights into the correlates of immune protection and pathogenesis. We sought to define these effects in patients with active TB by transcriptional profiling of tuberculin skin tests (TST) to make comprehensive molecular level assessments of in vivo human immune responses at the site of a standardised mycobacterial challenge. We showed that the TST transcriptome accurately reflects the molecular pathology at the site of human pulmonary TB, and used this approach to investigate immune dysregulation in HIV-1/TB co-infected patients with distinct clinical phenotypes associated with TST reactivity or anergy and unmasking TB immune reconstitution inflammatory syndrome (IRIS) after initiation of ART. HIV-1 infected patients with positive TSTs exhibited preserved Th1 responses but deficient immunoregulatory IL10-inducible responses. Those with clinically negative TSTs revealed profound anergy of innate as well as adaptive immune responses, except for preservation of type 1 interferon activity, implicated in impaired anti-mycobacterial immunity. Patients with unmasking TB IRIS showed recovery of Th1 immunity to normal levels, but exaggerated Th2-associated responses specifically. These mechanisms of immune dysregulation were localised to the tissue microenvironment and not evident in peripheral blood. TST molecular profiling categorised different mechanisms of immunological dysfunction in HIV-1 infection beyond the effects on CD4 T cells, each associated with increased risk of TB disease and amenable to host-directed therapies.
- Published
- 2016
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20. Transcriptional Profiling of Endobronchial Ultrasound-Guided Lymph Node Samples Aids Diagnosis of Mediastinal Lymphadenopathy.
- Author
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Tomlinson GS, Thomas N, Chain BM, Best K, Simpson N, Hardavella G, Brown J, Bhowmik A, Navani N, Janes SM, Miller RF, and Noursadeghi M
- Subjects
- Adult, Aged, Aged, 80 and over, Bronchoscopy methods, Female, Humans, Lymphatic Diseases diagnosis, Lymphatic Diseases etiology, Male, Mediastinal Diseases diagnosis, Mediastinal Diseases etiology, Mediastinum, Middle Aged, ROC Curve, Sarcoidosis, Pulmonary complications, Sarcoidosis, Pulmonary diagnosis, Young Adult, Endoscopic Ultrasound-Guided Fine Needle Aspiration methods, Lymph Nodes pathology, Lymphatic Diseases genetics, Mediastinal Diseases genetics, RNA analysis, Sarcoidosis, Pulmonary genetics, Transcriptome
- Abstract
Background: Endobronchial ultrasound (EBUS)-guided biopsy is the mainstay for investigation of mediastinal lymphadenopathy for laboratory diagnosis of malignancy, sarcoidosis, or TB. However, improved methods for discriminating between TB and sarcoidosis and excluding malignancy are still needed. We sought to evaluate the role of genomewide transcriptional profiling to aid diagnostic processes in this setting., Methods: Mediastinal lymph node samples from 88 individuals were obtained by EBUS-guided aspiration for investigation of mediastinal lymphadenopathy and subjected to transcriptional profiling in addition to conventional laboratory assessments. Computational strategies were used to evaluate the potential for using the transcriptome to distinguish between diagnostic categories., Results: Molecular signatures associated with granulomas or neoplastic and metastatic processes were clearly discernible in granulomatous and malignant lymph node samples, respectively. Support vector machine (SVM) learning using differentially expressed genes showed excellent sensitivity and specificity profiles in receiver operating characteristic curve analysis with area under curve values > 0.9 for discriminating between granulomatous and nongranulomatous disease, TB and sarcoidosis, and between cancer and reactive lymphadenopathy. A two-step decision tree using SVM to distinguish granulomatous and nongranulomatous disease, then between TB and sarcoidosis in granulomatous cases, and between cancer and reactive lymphadenopathy in nongranulomatous cases, achieved > 90% specificity for each diagnosis and afforded greater sensitivity than existing tests to detect TB and cancer. In some diagnostically ambiguous cases, computational classification predicted granulomatous disease or cancer before pathologic abnormalities were evident., Conclusions: Machine learning analysis of transcriptional profiling in mediastinal lymphadenopathy may significantly improve the clinical utility of EBUS-guided biopsies., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
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21. Identification of Therapeutic Targets of Inflammatory Monocyte Recruitment to Modulate the Allogeneic Injury to Donor Cornea.
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Lapp T, Zaher SS, Haas CT, Becker DL, Thrasivoulou C, Chain BM, Larkin DF, and Noursadeghi M
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Aqueous Humor metabolism, Cell Movement, Child, Child, Preschool, Cytokines metabolism, Endothelium, Corneal immunology, Endothelium, Corneal metabolism, Female, Graft Rejection metabolism, Graft Rejection pathology, Humans, Male, Middle Aged, Transplantation, Homologous, Young Adult, Corneal Transplantation, Endothelium, Corneal pathology, Graft Rejection immunology, Monocytes cytology
- Abstract
Purpose: We sought to test the hypothesis that monocytes contribute to the immunopathogenesis of corneal allograft rejection and identify therapeutic targets to inhibit monocyte recruitment., Methods: Monocytes and proinflammatory mediators within anterior chamber samples during corneal graft rejection were quantified by flow cytometry and multiplex protein assays. Lipopolysaccharide or IFN-γ stimulation of monocyte-derived macrophages (MDMs) was used to generate inflammatory conditioned media (CoM). Corneal endothelial viability was tested by nuclear counting, connexin 43, and propidium iodide staining. Chemokine and chemokine receptor expression in monocytes and MDMs was assessed in microarray transcriptomic data. The role of chemokine pathways in monocyte migration across microvascular endothelium was tested in vitro by chemokine depletion or chemokine receptor inhibitors., Results: Inflammatory monocytes were significantly enriched in anterior chamber samples within 1 week of the onset of symptoms of corneal graft rejection. The MDM inflammatory CoM was cytopathic to transformed human corneal endothelia. This effect was also evident in endothelium of excised human cornea and increased in the presence of monocytes. Gene expression microarrays identified monocyte chemokine receptors and cognate chemokines in MDM inflammatory responses, which were also enriched in anterior chamber samples. Depletion of selected chemokines in MDM inflammatory CoM had no effect on monocyte transmigration across an endothelial blood-eye barrier, but selective chemokine receptor inhibition reduced monocyte recruitment significantly., Conclusions: We propose a role for inflammatory monocytes in endothelial cytotoxicity in corneal graft rejection. Therefore, targeting monocyte recruitment offers a putative novel strategy to reduce donor endothelial cell injury in survival of human corneal allografts.
- Published
- 2015
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22. Hybrid epidemics--a case study on computer worm conficker.
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Zhang C, Zhou S, and Chain BM
- Subjects
- Internet, Models, Theoretical, Computer Security, Epidemics
- Abstract
Conficker is a computer worm that erupted on the Internet in 2008. It is unique in combining three different spreading strategies: local probing, neighbourhood probing, and global probing. We propose a mathematical model that combines three modes of spreading: local, neighbourhood, and global, to capture the worm's spreading behaviour. The parameters of the model are inferred directly from network data obtained during the first day of the Conficker epidemic. The model is then used to explore the tradeoff between spreading modes in determining the worm's effectiveness. Our results show that the Conficker epidemic is an example of a critically hybrid epidemic, in which the different modes of spreading in isolation do not lead to successful epidemics. Such hybrid spreading strategies may be used beneficially to provide the most effective strategies for promulgating information across a large population. When used maliciously, however, they can present a dangerous challenge to current internet security protocols.
- Published
- 2015
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23. Optimizing hybrid spreading in metapopulations.
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Zhang C, Zhou S, Miller JC, Cox IJ, and Chain BM
- Subjects
- Disease Outbreaks, Disease Transmission, Infectious, Epidemics, Humans, Models, Theoretical, Communicable Diseases epidemiology, Communicable Diseases transmission
- Abstract
Epidemic spreading phenomena are ubiquitous in nature and society. Examples include the spreading of diseases, information, and computer viruses. Epidemics can spread by local spreading, where infected nodes can only infect a limited set of direct target nodes and global spreading, where an infected node can infect every other node. In reality, many epidemics spread using a hybrid mixture of both types of spreading. In this study we develop a theoretical framework for studying hybrid epidemics, and examine the optimum balance between spreading mechanisms in terms of achieving the maximum outbreak size. We show the existence of critically hybrid epidemics where neither spreading mechanism alone can cause a noticeable spread but a combination of the two spreading mechanisms would produce an enormous outbreak. Our results provide new strategies for maximising beneficial epidemics and estimating the worst outcome of damaging hybrid epidemics.
- Published
- 2015
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24. Hybrid spreading mechanisms and T cell activation shape the dynamics of HIV-1 infection.
- Author
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Zhang C, Zhou S, Groppelli E, Pellegrino P, Williams I, Borrow P, Chain BM, and Jolly C
- Subjects
- Computational Biology, Female, HIV Infections virology, Humans, Male, CD4-Positive T-Lymphocytes immunology, HIV Infections immunology, HIV-1 immunology, Host-Pathogen Interactions immunology, Models, Immunological
- Abstract
HIV-1 can disseminate between susceptible cells by two mechanisms: cell-free infection following fluid-phase diffusion of virions and by highly-efficient direct cell-to-cell transmission at immune cell contacts. The contribution of this hybrid spreading mechanism, which is also a characteristic of some important computer worm outbreaks, to HIV-1 progression in vivo remains unknown. Here we present a new mathematical model that explicitly incorporates the ability of HIV-1 to use hybrid spreading mechanisms and evaluate the consequences for HIV-1 pathogenenesis. The model captures the major phases of the HIV-1 infection course of a cohort of treatment naive patients and also accurately predicts the results of the Short Pulse Anti-Retroviral Therapy at Seroconversion (SPARTAC) trial. Using this model we find that hybrid spreading is critical to seed and establish infection, and that cell-to-cell spread and increased CD4+ T cell activation are important for HIV-1 progression. Notably, the model predicts that cell-to-cell spread becomes increasingly effective as infection progresses and thus may present a considerable treatment barrier. Deriving predictions of various treatments' influence on HIV-1 progression highlights the importance of earlier intervention and suggests that treatments effectively targeting cell-to-cell HIV-1 spread can delay progression to AIDS. This study suggests that hybrid spreading is a fundamental feature of HIV infection, and provides the mathematical framework incorporating this feature with which to evaluate future therapeutic strategies.
- Published
- 2015
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25. Erratum to: Neutrophil Myeloperoxidase: Soldier and Statesman.
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Prokopowicz Z, Marcinkiewicz J, Katz DR, and Chain BM
- Published
- 2015
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26. Regulation of CYP27B1 and CYP24A1 hydroxylases limits cell-autonomous activation of vitamin D in dendritic cells.
- Author
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Kundu R, Chain BM, Coussens AK, Khoo B, and Noursadeghi M
- Subjects
- Dendritic Cells cytology, Female, Homeostasis physiology, Humans, Macrophages cytology, Macrophages immunology, Male, Monocytes cytology, Monocytes immunology, T-Lymphocytes cytology, Vitamin D3 24-Hydroxylase, 25-Hydroxyvitamin D3 1-alpha-Hydroxylase immunology, Calcitriol immunology, Dendritic Cells immunology, Lymphocyte Activation physiology, Steroid Hydroxylases immunology, T-Lymphocytes immunology
- Abstract
The active vitamin D metabolite 1α,25-dihydroxyvitamin D (1,25[OH]₂ D) potently inhibits DC priming of T-cell activation, suggesting that it mediates a homeostatic role in this context. Therefore, careful regulation of 1,25[OH]₂ D levels is necessary to avoid inappropriate inhibition of T-cell activation. Cell-autonomous control of vitamin D activity can be modulated by the action of the vitamin D-activating and -inactivating hydroxylases, CYP27B1, and CYP24A1, respectively. We show that in comparison to macrophages, human monocyte-derived DCs exhibit significantly less activation of 25-dihydroxyvitamin D to 1,25[OH]₂ D, and that DCs predominantly express a truncated CYP27B1 transcript that may contribute to the deficiency in activation of vitamin D. Furthermore, in response to stimulation with 1,25[OH]₂ D, upregulation of the inactivating enzyme CYP24A1 curtailed the functional effects of vitamin D in DCs, but not macrophages. Production of 1,25[OH]₂ D by macrophages was adequate to induce expression of vitamin D-responsive genes by DCs, inhibit DC maturation in response to innate immune stimulation and DC-dependent T-cell responses. Our data suggest that in comparison to macrophages, differential regulation of hydroxylases limits autocrine vitamin D activity in DCs, and that paracrine activation of vitamin D exerts a more potent mechanism for homeostatic control of DC function., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
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27. HIV-1 infection of macrophages dysregulates innate immune responses to Mycobacterium tuberculosis by inhibition of interleukin-10.
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Tomlinson GS, Bell LC, Walker NF, Tsang J, Brown JS, Breen R, Lipman M, Katz DR, Miller RF, Chain BM, Elkington PT, and Noursadeghi M
- Subjects
- Cells, Cultured, HIV Infections complications, Host-Pathogen Interactions, Humans, Immunosuppression Therapy, Macrophages microbiology, Macrophages virology, Tuberculosis, Pulmonary complications, HIV Infections immunology, HIV-1 immunology, Immunity, Innate, Interleukin-10 antagonists & inhibitors, Macrophages immunology, Mycobacterium tuberculosis immunology, Tuberculosis, Pulmonary immunology
- Abstract
Human immunodeficiency virus (HIV)-1 and Mycobacterium tuberculosis (M. tuberculosis) both target macrophages, which are key cells in inflammatory responses and their resolution. Therefore, we tested the hypothesis that HIV-1 may modulate macrophage responses to coinfection with M. tuberculosis. HIV-1 caused exaggerated proinflammatory responses to M. tuberculosis that supported enhanced virus replication, and were associated with deficient stimulus-specific induction of anti-inflammatory interleukin (IL)-10 and attenuation of mitogen-activated kinase signaling downstream of Toll-like receptor 2 and dectin-1 stimulation. Our in vitro data were mirrored by lower IL-10 and higher proinflammatory IL-1β in airway samples from HIV-1-infected patients with pulmonary tuberculosis compared with those with non-tuberculous respiratory tract infections. Single-round infection of macrophages with HIV-1 was sufficient to attenuate IL-10 responses, and antiretroviral treatment of replicative virus did not affect this phenotype. We propose that deficient homeostatic IL-10 responses may contribute to the immunopathogenesis of active tuberculosis and propagation of virus infection in HIV-1/M. tuberculosis coinfection.
- Published
- 2014
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28. Coupled IL-2-dependent extracellular feedbacks govern two distinct consecutive phases of CD4 T cell activation.
- Author
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Waysbort N, Russ D, Chain BM, and Friedman N
- Subjects
- Amino Acid Sequence, Animals, Antigen Presentation, Cells, Cultured, Enzyme-Linked Immunosorbent Assay, Flow Cytometry methods, Gene Expression Regulation immunology, Interleukin-2 biosynthesis, Interleukin-2 genetics, Interleukin-2 metabolism, Interleukin-2 Receptor alpha Subunit biosynthesis, Interleukin-2 Receptor alpha Subunit genetics, Mice, Mice, Inbred C3H, Mice, Transgenic, Molecular Sequence Data, Real-Time Polymerase Chain Reaction, Receptors, Antigen, T-Cell immunology, Robotics, Time Factors, CD4-Positive T-Lymphocytes immunology, Feedback, Physiological physiology, Interleukin-2 physiology, Lymphocyte Activation immunology
- Abstract
T cells integrate cell-specific Ag receptor signaling with shared signals mediated by secreted cytokines, which often involve regulatory feedback loops. IL-2 signaling, for example, reduces the synthesis of IL-2 and increases the synthesis of IL-2Rα-chain, whereas both genes require TCR signaling for their activation. The ways by which T cells dynamically integrate these private and public signals during activation are not well understood. We combined robotics, multiparameter flow cytometry, and real-time quantitative PCR to analyze T cell activation at high temporal resolution over several days. Two distinct temporal phases of T cell activation were evident. First, Ag-dependent signals activated low IL-2Rα and high IL-2 production, independent of IL-2 signaling. Subsequently, secreted IL-2 acted as a shared resource driving high IL-2Rα expression, reduced IL-2 synthesis, and cell proliferation. This transition was independent of continued TCR signaling. Our data allowed the determination of the parameters of the IL-2-mediated extracellular positive and negative feedback circuits and demonstrated that the two loops are coupled and become activated at a similar level of IL-2 signaling. We propose that temporal separation of private and shared signals allows T cells to first integrate Ag-specific responses and subsequently share information leading to collective decision making.
- Published
- 2013
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29. Importin-7 mediates nuclear trafficking of DNA in mammalian cells.
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Dhanoya A, Wang T, Keshavarz-Moore E, Fassati A, and Chain BM
- Subjects
- Active Transport, Cell Nucleus drug effects, Active Transport, Cell Nucleus genetics, HeLa Cells, Heterogeneous-Nuclear Ribonucleoprotein Group A-B genetics, Heterogeneous-Nuclear Ribonucleoprotein Group A-B metabolism, Humans, Immunity, Innate, Interferons metabolism, Karyopherins genetics, Maltose-Binding Proteins genetics, Maltose-Binding Proteins metabolism, Nuclear Localization Signals genetics, Nuclear Localization Signals metabolism, Peptide Fragments genetics, Peptide Fragments metabolism, Polylysine pharmacology, Protein Sorting Signals genetics, Receptors, Cytoplasmic and Nuclear genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Cell Nucleus metabolism, DNA, Mitochondrial metabolism, DNA, Superhelical metabolism, Karyopherins metabolism, Receptors, Cytoplasmic and Nuclear metabolism
- Abstract
Eukaryotic cells have the ability to uptake and transport endogenous and exogenous DNA in their nuclei, however little is known about the specific pathways involved. Here we show that the nuclear transport receptor importin 7 (imp7) supports nuclear import of supercoiled plasmid DNA and human mitochondrial DNA in a Ran and energy-dependent way. The imp7-dependent pathway was specifically competed by excess DNA but not by excess of maltose-binding protein fused with the classical nuclear localizing signal (NLS) or the M9 peptides. Transport of DNA molecules complexed with poly-l-lysine was impaired in intact cells depleted of imp7, and DNA complexes remained localized in the cytoplasm. Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls. Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response. Our results provide evidence that imp7 is a non-redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response., (© 2012 John Wiley & Sons A/S.)
- Published
- 2013
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30. Role of DNA topology in uptake of polyplex molecules by dendritic cells.
- Author
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Dhanoya A, Chain BM, and Keshavarz-Moore E
- Subjects
- Cells, Cultured, DNA pharmacokinetics, DNA, Superhelical chemistry, DNA, Superhelical pharmacokinetics, Dendritic Cells immunology, Genetic Vectors pharmacokinetics, Humans, Polylysine chemistry, Vaccines, DNA chemistry, Vaccines, DNA pharmacokinetics, DNA chemistry, Dendritic Cells metabolism, Genetic Vectors chemistry, Polylysine pharmacokinetics, Transfection methods
- Abstract
Dendritic cells (DCs) are an attractive target for DNA vaccines as they are potent antigen presenting cells. This study demonstrated how non-viral gene delivery to DCs involving complexes of poly-l-lysine (PLL) and plasmid DNA (pDNA) (polyplexes) showed dependence on DNA vector topology. DNA topology is of importance from both production and regulatory viewpoints. In our previous study with CHO cells we demonstrated that polyplex uptake was dependent on DNA topology whereby complexes containing supercoiled (SC) pDNA were smaller, more resistant to nucleases and more effectively condensed by PLL than open circular (OC) and linear-pDNA complexes. In this study polyplex uptake in DCs was measured qualitatively and quantitatively by confocal microscopy along with gene expression studies and measurement of DC phenotype. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Quantification studies revealed that by 1h following uptake 15% (±2.59% relative standard error [RSE]) of SC-pDNA polyplexes were identified to be associated (fluorescent co-localisation) with the nucleus, in comparison to no nuclear association identified for OC- and linear-pDNA complexes. By 48 h following uptake, 30% (±1.82% RSE) of SC-pDNA complexes associated with the nucleus in comparison to 16% (±4.40% RSE) and 12% (±6.97% RSE) of OC- and linear-pDNA polyplexes respectively. Confocal microscopy images showed how DNA and PLL remained associated following uptake by dual labelling. Polyplex (containing 20 μg pDNA) gene expression (plasmid encoded lacZ [β-galactosidase] reporter gene) in DCs was greatest for SC-pDNA polyplexes at 14.12% unlike that of OC- (9.59%) and linear-pDNA (7.43%). DCs express cell surface markers which contribute towards antigen presentation. Polyplex gene expression did not alter DC phenotype through surface marker expression. This may be due to the pDNA dose employed (20μg) as other studies have used doses as high as 200 μg pDNA to induce DC phenotypic changes. Although no change in DC phenotype occurred, this could be advantageous in terms of biocompatibility. Collectively these results indicate that DNA topology is an important parameter for DC vector design, particularly pDNA in the SC conformation in regards to DNA vaccination studies., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
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31. Neutrophil myeloperoxidase: soldier and statesman.
- Author
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Prokopowicz Z, Marcinkiewicz J, Katz DR, and Chain BM
- Subjects
- Adaptive Immunity immunology, Animals, Humans, Immunity, Innate immunology, Inflammation immunology, Inflammation physiopathology, Oxidation-Reduction, Reactive Oxygen Species metabolism, Tissue Distribution, Neutrophils enzymology, Peroxidase metabolism
- Abstract
Myeloperoxidase (MPO) is a major protein constituent of the primary granules of vertebrate neutrophils. It catalyses the hydrogen peroxide-mediated oxidation of halide ions to hypohalous acids, especially HOCl. These reactive oxygen species can participate in a variety of secondary reactions, leading to modifications of amino acids and many types of biological macromolecules. The classic paradigm views MPO as a component of the phagocyte oxygen-dependent intracellular microbicidal system, and thus an important arm of the effector phase of innate immune responses. However, the limited immunodeficiency associated with lack of MPO in mouse and human models has challenged this paradigm. In this review we examine more recent information on the interaction between MPO, its bioreactive reaction products, and targets within the inflammatory microenvironment. We propose that two assumptions of the current model may require revisiting. First, many important targets of MPO modification are extracellular, rather than present only within the phagolysosome, such as various components of neutrophil extracellular traps. Second, we suggest that the pro-inflammatory pathological role of MPO may be a particular feature of chronic inflammation. In the physiological setting of acute neutrophil-mediated inflammation MPO may also form part of a negative feedback loop which down-regulates inflammation, limits tissue damage, and facilitates the switch from innate to adaptive immunity. This different perspective on this well-studied enzyme may usefully inform further research into its function in health and disease.
- Published
- 2012
- Full Text
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32. Adherent human alveolar macrophages exhibit a transient pro-inflammatory profile that confounds responses to innate immune stimulation.
- Author
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Tomlinson GS, Booth H, Petit SJ, Potton E, Towers GJ, Miller RF, Chain BM, and Noursadeghi M
- Subjects
- Antigen Presentation drug effects, Antigen Presentation genetics, Cell Adhesion drug effects, Cell Adhesion genetics, Cell Separation, Cells, Cultured, Gene Expression Profiling, Genome, Human genetics, Histocompatibility Antigens Class II genetics, Humans, Immunity, Innate drug effects, Inflammation genetics, Lipopolysaccharides pharmacology, Macrophages, Alveolar drug effects, Phenotype, Time Factors, Transcription Factors metabolism, Up-Regulation drug effects, Up-Regulation genetics, Immunity, Innate immunology, Inflammation immunology, Inflammation pathology, Macrophages, Alveolar immunology, Macrophages, Alveolar pathology
- Abstract
Alveolar macrophages (AM) are thought to have a key role in the immunopathogenesis of respiratory diseases. We sought to test the hypothesis that human AM exhibit an anti-inflammatory bias by making genome-wide comparisons with monocyte derived macrophages (MDM). Adherent AM obtained by bronchoalveolar lavage of patients under investigation for haemoptysis, but found to have no respiratory pathology, were compared to MDM from healthy volunteers by whole genome transcriptional profiling before and after innate immune stimulation. We found that freshly isolated AM exhibited a marked pro-inflammatory transcriptional signature. High levels of basal pro-inflammatory gene expression gave the impression of attenuated responses to lipopolysaccharide (LPS) and the RNA analogue, poly IC, but in rested cells pro-inflammatory gene expression declined and transcriptional responsiveness to these stimuli was restored. In comparison to MDM, both freshly isolated and rested AM showed upregulation of MHC class II molecules. In most experimental paradigms ex vivo adherent AM are used immediately after isolation. Therefore, the confounding effects of their pro-inflammatory profile at baseline need careful consideration. Moreover, despite the prevailing view that AM have an anti-inflammatory bias, our data clearly show that they can adopt a striking pro-inflammatory phenotype, and may have greater capacity for presentation of exogenous antigens than MDM.
- Published
- 2012
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- View/download PDF
33. Chemical toxicity to keratinocytes triggers dendritic cell activation via an IL-1α path.
- Author
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Matjeka T, Summerfield V, Noursadeghi M, and Chain BM
- Subjects
- Dendritic Cells metabolism, Dinitrobenzenes toxicity, Host-Pathogen Interactions immunology, Humans, Keratinocytes metabolism, Dendritic Cells immunology, Interleukin-1alpha metabolism, Keratinocytes drug effects, Signal Transduction
- Published
- 2012
- Full Text
- View/download PDF
34. Transcriptional profiling of innate and adaptive human immune responses to mycobacteria in the tuberculin skin test.
- Author
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Tomlinson GS, Cashmore TJ, Elkington PT, Yates J, Lehloenya RJ, Tsang J, Brown M, Miller RF, Dheda K, Katz DR, Chain BM, and Noursadeghi M
- Subjects
- Humans, Hypersensitivity, Delayed immunology, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction, T-Lymphocytes, Cytotoxic immunology, Th1 Cells immunology, Tuberculosis immunology, Adaptive Immunity genetics, Gene Expression Profiling, Hypersensitivity, Delayed genetics, Immunity, Innate genetics, Mycobacterium immunology, Tuberculin Test
- Abstract
The tuberculin skin test (TST) is a model of integrated innate and adaptive human immune responses to Mycobacterium tuberculosis, but the component processes that are involved in this model have not previously been defined in vivo. We used transcriptional profiling to study these responses within the TST at molecular and system levels. Skin biopsies from TST injection sites were examined in subjects classified as TST(+) or TST(-) by clinical and histological criteria. Genome-wide expression arrays showed evolution of immune responses reflecting T-cell activation and recruitment with uniquely Th1-polarized responses and cytotoxic T cells (CTLs). In addition, distinct innate immune and IFN-γ-stimulated gene expression signatures were identified, under the regulation of NF-κB and STAT1 transcriptional control. These were highly enriched for chemokines and MHC class II molecules providing a potential mechanism for paracrine amplification of inflammatory responses in the TST, by supporting cellular recruitment and enhancing antigen presentation. The same repertoire of innate and adaptive immune responses was evident in TST(+) and TST(-) subjects alike, clinically positive TSTs being distinguished only by quantitatively greater differences. These data provide new insights into complex multifaceted responses within the TST, with much greater sensitivity than previous clinical or histological assessments., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2011
- Full Text
- View/download PDF
35. The impact of DNA topology on polyplex uptake and transfection efficiency in mammalian cells.
- Author
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Dhanoya A, Chain BM, and Keshavarz-Moore E
- Subjects
- Animals, CHO Cells, Cricetinae, Cricetulus, DNA, Superhelical chemistry, DNA, Superhelical genetics, Genetic Vectors, Plasmids chemistry, Plasmids genetics, DNA chemistry, DNA genetics, Polylysine chemistry, Transfection methods
- Abstract
The effect of DNA vector topology when complexed to poly-l-lysine (PLL) and its quantification in transfection efficiency has not been fully addressed even though it is thought to be of importance from both production and regulatory viewpoints. This study investigates and quantifies cell uptake followed by transfection efficiency of PLL:DNA complexes (polyplexes) in Chinese hamster ovary (CHO) cells and their dependence on DNA topology. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Characterization of PLL conjugated to a 6.9kb plasmid was carried out. Dual labeling of both the plasmid DNA (pDNA) and PLL enabled quantitative tracking of the complexed as well as dissociated elements, within the cell, and their dependence on DNA topology. Polyplex uptake was quantified by confocal microscopy and image analysis. Supercoiled (SC) pDNA when complexed with PLL, forms a polyplex with a mean diameter of 139.06nm (±0.84% relative standard error [RSE]), whereas open circular (OC) and linear-pDNA counterparts displayed mean diameters of 305.54 (±3.2% RSE) and 841.5nm (±7.2% RSE) respectively. Complexes containing SC-pDNA were also more resistant to nuclease attack than its topological counterparts. Confocal microscope images reveal how the PLL and DNA remain bound post transfection. Quantification studies revealed that by 1h post transfection 61% of SC-pDNA polyplexes were identified to be associated with the nucleus, in comparison to OC- (24.3%) and linear-pDNA polyplexes (3.5%) respectively. SC-pDNA polyplexes displayed the greatest transfection efficiency of 41% which dwarfed that of linear-pDNA polyplexes of 18.6%. Collectively these findings emphasize the importance of pDNA topology when complexed with PLL for gene delivery with the SC-form being a key pre-requisite., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2011
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36. DC priming by M. vaccae inhibits Th2 responses in contrast to specific TLR2 priming and is associated with selective activation of the CREB pathway.
- Author
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Le Bert N, Chain BM, Rook G, and Noursadeghi M
- Subjects
- Animals, Cytokines metabolism, Dendritic Cells cytology, Dendritic Cells metabolism, HEK293 Cells, Humans, Immunity, Innate genetics, Mice, Monocytes cytology, Th2 Cells cytology, Th2 Cells metabolism, Th2 Cells microbiology, Transcription, Genetic immunology, Cyclic AMP Response Element-Binding Protein metabolism, Dendritic Cells immunology, Dendritic Cells microbiology, Mycobacterium immunology, Signal Transduction immunology, Th2 Cells immunology, Toll-Like Receptor 2 metabolism
- Abstract
The environmental mycobacterium, M. vaccae has been used in mouse models to support the contemporary hygiene hypothesis that non-pathogenic microorganisms reduce allergy associated T helper (Th)2 responses and inflammatory diseases by augmenting regulatory T cells. However, data for human models and possible mechanisms are limited. We tested the effect of innate immune interactions between human DC and M. vaccae on DC-dependent T cell responses. M. vaccae activation of DC via Toll like receptor (TLR)2 was compared to a specific TLR2 ligand (Pam(3)CSK4) and alternative stimulation with a TLR4 ligand (LPS). M. vaccae induced DC dependent inhibition of Th2 responses, in contrast to Pam(3)CSK4, which had the opposite effect and LPS, which had no polarizing effect. DC maturation, gene expression and cytokine production, in response to each stimulus did not correlate with the specific functional effects. Comparable DC transcriptional responses to M. vaccae and Pam(3)CSK4 suggested that TLR2 mediated transcriptional regulation was not sufficient for inhibition of Th2 responses. Transcription factor enrichment analysis and assessment of signaling events, implicated a role for selective early activation of the CREB pathway by M. vaccae. Further study of the CREB pathway may provide novel insight into the molecular mechanisms of DC-dependent T cell polarization.
- Published
- 2011
- Full Text
- View/download PDF
37. Differential requirement for cathepsin D for processing of the full length and C-terminal fragment of the malaria antigen MSP1.
- Author
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Tulone C, Sponaas AM, Raiber EA, Tabor AB, Langhorne J, and Chain BM
- Subjects
- Animals, Antibody Formation drug effects, Antibody Formation immunology, Antigen Presentation drug effects, Bone Marrow Cells cytology, Bone Marrow Cells drug effects, Bone Marrow Cells immunology, Cathepsin D deficiency, Chimera immunology, Dendritic Cells drug effects, Dendritic Cells immunology, Erythrocytes drug effects, Erythrocytes parasitology, Histocompatibility Antigens Class II immunology, Immunoglobulin G immunology, Malaria parasitology, Merozoites drug effects, Merozoites immunology, Mice, Parasitemia immunology, Parasitemia parasitology, Phenotype, Plasmodium chabaudi drug effects, Plasmodium chabaudi immunology, Protease Inhibitors pharmacology, Spleen drug effects, Spleen immunology, Spleen parasitology, Antigen Presentation immunology, Antigens, Protozoan chemistry, Antigens, Protozoan immunology, Cathepsin D metabolism, Malaria immunology, Merozoite Surface Protein 1 chemistry, Merozoite Surface Protein 1 immunology
- Abstract
Merozoite Surface Protein 1 is expressed on the surface of malaria merozoites and is important for invasion of the malaria parasite into erythrocytes. MSP1-specific CD4 T cell responses and antibody can confer protective immunity in experimental models of malaria. In this study we explore the contributions of cathepsins D and E, two aspartic proteinases previously implicated in antigen processing, to generating MSP1 CD4 T-cell epitopes for presentation. The absence of cathepsin D, a late endosome/lysosomal enzyme, is associated with a reduced presentation of MSP1 both following in vitro processing of the epitope MSP1 from infected erythrocytes by bone marrow-derived dendritic cells, and following in vivo processing by splenic CD11c+ dendritic cells. By contrast, processing and presentation of the soluble recombinant protein fragment of MSP1 is unaffected by the absence of cathepsin D, but is inhibited when both cathepsin D and E are absent. The role of different proteinases in generating the CD4 T cell repertoire, therefore, depends on the context in which an antigen is introduced to the immune system.
- Published
- 2011
- Full Text
- View/download PDF
38. A quantitative proteomics design for systematic identification of protease cleavage events.
- Author
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Impens F, Colaert N, Helsens K, Ghesquière B, Timmerman E, De Bock PJ, Chain BM, Vandekerckhove J, and Gevaert K
- Subjects
- Amino Acid Sequence, Animals, Cathepsin D metabolism, Cathepsin E metabolism, Humans, Hydrolysis, Mice, Molecular Sequence Data, Substrate Specificity, Caspase 3 metabolism, Proteomics
- Abstract
We present here a novel proteomics design for systematic identification of protease cleavage events by quantitative N-terminal proteomics, circumventing the need for time-consuming manual validation. We bypass the singleton detection problem of protease-generated neo-N-terminal peptides by introducing differential isotopic proteome labeling such that these substrate reporter peptides are readily distinguished from all other N-terminal peptides. Our approach was validated using the canonical human caspase-3 protease and further applied to mouse cathepsin D and E substrate processing in a mouse dendritic cell proteome, identifying the largest set of protein protease substrates ever reported and gaining novel insight into substrate specificity differences of these cathepsins.
- Published
- 2010
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39. Targeted delivery of antigen processing inhibitors to antigen presenting cells via mannose receptors.
- Author
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Raiber EA, Tulone C, Zhang Y, Martinez-Pomares L, Steed E, Sponaas AM, Langhorne J, Noursadeghi M, Chain BM, and Tabor AB
- Subjects
- Animals, Antigen Presentation immunology, Cathepsin D antagonists & inhibitors, Dendritic Cells metabolism, Epitopes, T-Lymphocyte metabolism, Immunoprecipitation, Lectins, C-Type metabolism, Macrophages, Peritoneal immunology, Macrophages, Peritoneal metabolism, Mannose chemistry, Mannose metabolism, Mannose Receptor, Mannose-Binding Lectins metabolism, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Transgenic, Protease Inhibitors chemical synthesis, Protease Inhibitors pharmacology, Rabbits, Receptors, Cell Surface metabolism, Spleen cytology, Spleen immunology, Spleen metabolism, Antigen Presentation drug effects, Aspartic Acid Endopeptidases antagonists & inhibitors, Aspartic Acid Endopeptidases metabolism, Dendritic Cells drug effects, Dendritic Cells immunology, Pepstatins chemical synthesis, Pepstatins immunology, Pepstatins pharmacology
- Abstract
Improved chemical inhibitors are required to dissect the role of specific antigen processing enzymes and to complement genetic models. In this study we explore the in vitro and in vivo properties of a novel class of targeted inhibitor of aspartic proteinases, in which pepstatin is coupled to mannosylated albumin (MPC6), creating an inhibitor with improved solubility and the potential for selective cell tropism. Using these compounds, we have demonstrated that MPC6 is taken up via mannose receptor facilitated endocytosis, leading to a slow but continuous accumulation of inhibitor within large endocytic vesicles within dendritic cells and a parallel inhibition of intracellular aspartic proteinase activity. Inhibition of intracellular proteinase activity is associated with reduction in antigen processing activity, but this is epitope-specific, preferentially inhibiting processing of T cell epitopes buried within compact proteinase-resistant protein domains. Unexpectedly, we have also demonstrated, using quenched fluorescent substrates, that little or no cleavage of the disulfide linker takes place within dendritic cells. This does not appear to affect the activity of MPC6 as an inhibitor of cathepsins D and E in vitro and in vivo. Finally, we have shown that MPC6 selectively targets dendritic cells and macrophages in spleen in vivo. Preliminary results suggest that access to nonlymphoid tissues is very limited in the steady state but is strongly enhanced at local sites of inflammation. The strategy adopted for MPC6 synthesis may therefore represent a more general way to deliver chemical inhibitors to cells of the innate immune system, especially at sites of inflammation.
- Published
- 2010
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- View/download PDF
40. Src-mediated regulation of inflammatory responses by actin polymerization.
- Author
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Kim JY, Lee YG, Kim MY, Byeon SE, Rhee MH, Park J, Katz DR, Chain BM, and Cho JY
- Subjects
- Actins pharmacology, Animals, Cell Line, Cells, Cultured, Cytochalasin B pharmacology, Humans, Inflammation enzymology, Inflammation pathology, Male, Mice, Mice, Inbred C57BL, Polymers pharmacology, Actins metabolism, Inflammation Mediators physiology, Polymers metabolism, src-Family Kinases physiology
- Abstract
Although the role of the actin cytoskeleton has become increasingly elucidated, the role of actin polymerization in inflammatory processes remains poorly understood. Here, we examine the role of the actin cytoskeleton during LPS-mediated inflammatory events in RAW264.7 cells and peritoneal macrophages. We observed that actin cytoskeleton disruption by cytochalasin B and siRNA to cytoplasmic actin strongly down-regulated LPS-mediated inflammatory responses such as NO production, PGE(2) release, and TNF-alpha secretion. Actin cytoskeleton disruption by cytochalasin B down-regulated a series of signaling cascades including PI3K, Akt, and IKK, but not MAPKs, necessary for NF-kappaB activation without down-regulating total forms of the proteins as assessed by measuring their phosphorylation levels. In particular, cytochalasin B significantly inhibited LPS-induced both phosphorylation and kinase activity of Src without altering total level, implying that Src may be a potential pharmacological target of actin cytoskeleton rearrangement. Moreover, the direct association of Src with actin was actin polymerization-dependent according to immunoprecipitation analysis performed with a GFP-actin wild type and HA-tagged Src. Therefore, our data suggest that actin cytoskeleton rearrangement may be a key event during the regulation of inflammatory responses that modulates the activity of Src and its downstream signaling molecules.
- Published
- 2010
- Full Text
- View/download PDF
41. Hypochlorous acid: a natural adjuvant that facilitates antigen processing, cross-priming, and the induction of adaptive immunity.
- Author
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Prokopowicz ZM, Arce F, Biedroń R, Chiang CL, Ciszek M, Katz DR, Nowakowska M, Zapotoczny S, Marcinkiewicz J, and Chain BM
- Subjects
- Adjuvants, Immunologic pharmacology, Animals, Antigen-Presenting Cells immunology, Granulocytes, Histocompatibility Antigens Class II, Immunity, Innate, Mice, Ovalbumin immunology, Receptors, LDL metabolism, T-Lymphocytes immunology, Adaptive Immunity drug effects, Antigen Presentation drug effects, Cross-Priming drug effects, Hypochlorous Acid pharmacology
- Abstract
The production of hypochlorous acid (HOCl) is a characteristic of granulocyte activation, a hallmark of the early phase of innate immune responses. In this study, we show that, in addition to its well-established role as a microbicide, HOCl can act as a natural adjuvant of adaptive immunity. HOCl enhances the T cell responses to the model Ag OVA, facilitating the processing and presentation of this protein via the class II MHC pathway. HOCl modification also enhances cross-presentation of the tumor Ag tyrosinase-related protein 2 via class I MHC. The adjuvant effects of HOCl are independent of TLR signaling. The enhanced presentation of HOCl-modified OVA is mediated via modification of the N-linked carbohydrate side chain rather than formation of protein aldehydes or chloramines. HOCl-modified OVA is taken up more efficiently by APCs and is degraded more efficiently by proteinases. Atomic force microscopy demonstrated that enhanced uptake is mediated via specific receptor binding, one candidate for which is the scavenger receptor lectin-like oxidized low-density lipoprotein receptor, which shows enhanced binding to chlorinated OVA. A function of HOCl is therefore to target glycoprotein Ags to scavenger receptors on the APC surface. This additional mechanism linking innate and adaptive immunity suggests novel strategies to enhance immunity to vaccines.
- Published
- 2010
- Full Text
- View/download PDF
42. Conventional protein kinase C plays a critical role in negative regulation of CD98-induced homotypic aggregation.
- Author
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Cho JY, Katz DR, Skubitz KM, and Chain BM
- Subjects
- Adenosine Triphosphate metabolism, Binding Sites, Calcium metabolism, Cell Aggregation drug effects, Diglycerides metabolism, Down-Regulation drug effects, Humans, Isoenzymes metabolism, Protein Binding, Protein Transport, Signal Transduction drug effects, Tetradecanoylphorbol Acetate analogs & derivatives, Tetradecanoylphorbol Acetate pharmacology, U937 Cells, Cell Aggregation physiology, Fusion Regulatory Protein-1 metabolism, Protein Kinase C metabolism
- Abstract
CD98, a heterodimeric type II transmembrane protein, is involved in many different cellular events, ranging from amino acid transport to cell-cell adhesion. Little is known about the positive and negative signalling pathways involved in these responses. Therefore, we examined the role of conventional protein kinase C (PKC) isoforms during CD98-induced intracellular signalling and homotypic aggregation of U937 cells. The CD98-induced aggregation was enhanced by the general protein kinase inhibitors GF109203X and staurosporin, and by specific PKC-alpha/-beta peptide inhibitor 19-27, but inhibited by PKC activators such as phorbol 12-myristate 13-acetate (PMA). PMA-inhibition was reversed by PKC inhibitors recognising the ATP-binding site in PKC (e.g. staurosporin, GF109203X and Go6983). Inhibitors which bind to diacylglycerol (DAG) or Ca(2+)-binding sites of PKC (calphostin C and Go6967) had no effect. PMA-induced translocation of conventional PKC (cPKC) isozymes (alpha, beta and gamma), but decreased the expression of PKC-delta, which plays an important role in CD98-induced homotypic aggregation. PMA treatment also suppressed the surface level of CD98 but not CD29, CD18 and CD147, dose- and time-dependently. These data provide evidence that PMA-responsive cPKC isoforms (alpha, beta and gamma) play a key role in negative regulation of CD98 signalling and homotypic aggregation.
- Published
- 2010
- Full Text
- View/download PDF
43. HIV-1 infection of macrophages is dependent on evasion of innate immune cellular activation.
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Tsang J, Chain BM, Miller RF, Webb BL, Barclay W, Towers GJ, Katz DR, and Noursadeghi M
- Subjects
- Cells, Cultured, Gene Expression Regulation, Viral, HIV Infections genetics, HIV Infections virology, Humans, Immune Evasion immunology, Immunity, Cellular immunology, Immunity, Innate genetics, Macrophage Activation genetics, Macrophage Activation immunology, Macrophages immunology, NF-kappa B metabolism, Signal Transduction genetics, Signal Transduction immunology, Viral Tropism, Virus Replication genetics, HIV Infections immunology, HIV-1 physiology, Immunity, Innate immunology, Macrophages virology, Virus Replication immunology
- Abstract
Objective: The cellular innate immune response to HIV-1 is poorly characterized. In view of HIV-1 tropism for macrophages, which can be activated via pattern recognition receptors to trigger antimicrobial defences, we investigated innate immune responses to HIV-1 by monocyte-derived macrophages., Design: In a model of productive HIV-1 infection, cellular innate immune responses to HIV-1 were investigated, at the level of transcription factor activation, specific gene expression and genome-wide transcriptional profiling. In addition, the viral determinants of macrophage responses and the physiological effect of innate immune cellular activation on HIV-1 replication were assessed., Results: Productive HIV-1 infection did not activate nuclear factor-kappaB and interferon regulatory factor 3 transcription factors or interferon gene expression (IFN) and caused remarkably small changes to the host-cell transcriptome, with no evidence of inflammatory or IFN signatures. Evasion of IFN induction was not dependent on HIV-1 envelope-mediated cellular entry, inhibition by accessory proteins or reverse transcription of ssRNA that may reduce innate immune cellular activation by viral RNA. Furthermore, IFNbeta priming did not sensitize responses to HIV-1. Importantly, exogenous IFNbeta or stimulation with the RNA analogue poly I:C to simulate innate immune activation invoked HIV-1 restriction., Conclusion: We conclude that macrophages lack functional pattern recognition receptors for this virus and that HIV-1 tropism for macrophages helps to establish a foothold in the host without triggering innate immune cellular activation, which would otherwise block viral infection effectively.
- Published
- 2009
- Full Text
- View/download PDF
44. Distinct role of spleen tyrosine kinase in the early phosphorylation of inhibitor of kappaB alpha via activation of the phosphoinositide-3-kinase and Akt pathways.
- Author
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Lee YG, Chain BM, and Cho JY
- Subjects
- Animals, CSK Tyrosine-Protein Kinase, Enzyme Activation, Gene Expression Regulation, Enzymologic, Intracellular Signaling Peptides and Proteins antagonists & inhibitors, Intracellular Signaling Peptides and Proteins genetics, Lipopolysaccharides pharmacology, Macrophages, Peritoneal drug effects, Macrophages, Peritoneal enzymology, Male, Mice, Mice, Inbred C57BL, Mice, Inbred ICR, NF-KappaB Inhibitor alpha, NF-kappa B metabolism, Phosphorylation, Protein-Tyrosine Kinases antagonists & inhibitors, Protein-Tyrosine Kinases genetics, RNA, Small Interfering genetics, Signal Transduction, Stilbenes pharmacology, Syk Kinase, Transfection, src-Family Kinases, I-kappa B Proteins metabolism, Intracellular Signaling Peptides and Proteins metabolism, Phosphatidylinositol 3-Kinases metabolism, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins c-akt metabolism
- Abstract
Nuclear factor (NF)-kappaB activation is a critical step in the triggering of inflammatory responses by macrophages. Although numerous investigations have been reported, the precise regulatory mechanisms controlling inflammatory responses mediated by NF-kappaB remain unclear. In this study, we investigated the early signaling events responsible for modulating NF-kappaB activation using various parameters, such as the expression of pro-inflammatory genes and the phosphorylation levels of inhibitor of kappaB alpha (IkappaB alpha) and its upstream kinases. Lipopolysaccharide (LPS) treatment biphasically induced activation of IkappaB alpha phosphorylation at 5 and 30 min, which induced subsequent pro-inflammatory gene expression that was maximized at 45 and 90 min. Of the intracellular signals tested, a series of signaling cascades composed of spleen tyrosine kinase (Syk), phosphoinositide-3-kinase (PI3K), and Akt (protein kinase B) were involved in regulating early phosphorylation of IkappaB alpha, according to biochemical and pharmacological analyses. Therefore, our data suggests that Syk-mediated activation of intracellular signaling in response to LPS may play an important role in LPS-induced inflammatory signaling events. Thus, Syk may be a potential target for the development of potent anti-inflammatory drugs.
- Published
- 2009
- Full Text
- View/download PDF
45. Genome-wide innate immune responses in HIV-1-infected macrophages are preserved despite attenuation of the NF-kappa B activation pathway.
- Author
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Noursadeghi M, Tsang J, Miller RF, Straschewski S, Kellam P, Chain BM, and Katz DR
- Subjects
- Cell Line, Cells, Cultured, Down-Regulation genetics, Down-Regulation immunology, Gene Expression Profiling, Gene Expression Regulation, Viral immunology, HLA-D Antigens biosynthesis, HLA-D Antigens genetics, HLA-D Antigens immunology, Humans, Lipopolysaccharides pharmacology, Macrophage Activation genetics, Macrophages metabolism, NF-kappa B metabolism, NF-kappa B physiology, Signal Transduction genetics, Transcription, Genetic immunology, Virus Latency genetics, Virus Latency immunology, Genome, Human immunology, HIV-1 immunology, Immunity, Innate genetics, Macrophage Activation immunology, Macrophages immunology, Macrophages virology, NF-kappa B antagonists & inhibitors, Signal Transduction immunology
- Abstract
Macrophages contribute to HIV-1 infection at many levels. They provide permissive cells at the site of inoculation, augment virus transfer to T cells, generate long-lived viral reservoirs, and cause bystander cell apoptosis. A body of evidence suggests that the role of macrophages in cellular host defense is also compromised by HIV-1 infection. In this respect, macrophages are potent cells of the innate immune system that initiate and regulate wide-ranging immunological responses. This study focuses on the effect of HIV-1 infection on innate immune responses by macrophages at the level of signal transduction, whole genome transcriptional profiling, and cytokine secretion. We show that in an ex vivo model, M-CSF-differentiated monocyte-derived macrophages uniformly infected with replicating CCR5-tropic HIV-1, without cytopathic effect, exhibit selective attenuation of the NF-kappaB activation pathway in response to TLR4 and TLR2 stimulation. However, functional annotation clustering analysis of genome-wide transcriptional responses to LPS stimulation suggests substantial preservation of gene expression changes at the systems level, with modest attenuation of a subset of up-regulated LPS-responsive genes, and no effect on a selection of inflammatory cytokine responses at the protein level. These results extend existing reports of inhibitory interactions between HIV-1 accessory proteins and NF-kappaB signaling pathways, and whole genome expression profiling provides comprehensive assessment of the consequent effects on immune response gene expression. Unexpectedly, our data suggest innate immune responses are broadly preserved with limited exceptions, and pave the way for further study of the complex relationship between HIV-1 and immunological pathways within macrophages.
- Published
- 2009
- Full Text
- View/download PDF
46. HIV blocking antibodies following immunisation with chimaeric peptides coding a short N-terminal sequence of the CCR5 receptor.
- Author
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Chain BM, Noursadeghi M, Gardener M, Tsang J, and Wright E
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Blocking blood, Cell Line, Tumor, Cells, Cultured, Epitopes, T-Lymphocyte immunology, HIV Antibodies blood, HIV Infections prevention & control, HIV-1, Humans, Immunization, Macrophages, Molecular Sequence Data, Peptides chemistry, Peptides genetics, Peptides metabolism, Rabbits, Receptors, CCR5 genetics, Receptors, CCR5 metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Tetanus Toxoid genetics, Tetanus Toxoid metabolism, Peptides immunology, Receptors, CCR5 chemistry, Receptors, CCR5 immunology, Recombinant Fusion Proteins immunology, Tetanus Toxoid immunology
- Abstract
The chemokine receptor CCR5 is required for cellular entry by many strains of HIV, and provides a potential target for molecules, including antibodies, designed to block HIV transmission. This study investigates a novel approach to stimulate antibodies to CCR5. Rabbits were immunised with chimaeric peptides which encode a short fragment of the N-terminal sequence of CCR5, as well as an unrelated T cell epitope from Tetanus toxoid. Immunisation with these chimaeric peptides generates a strong antibody response which is highly focused on the N-terminal CCR5 sequence. The antibody to the chimaeric peptide containing an N-terminal methionine also recognises the full length CCR5 receptor on the cell surface, albeit at higher concentrations. Further comparison of binding to intact CCR5 with binding to CCR5 peptide suggest that the receptor specific antibody generated represents a very small fragment of the total anti-peptide antibody. These findings are consistent with the hypothesis that the N-terminal peptide in the context of the intact receptor has a different structure to that of the synthetic peptide. Finally, the antibody was able to block HIV infection of macrophages in vitro. Thus results of this study suggest that N-terminal fragments of CCR5 may provide potential immunogens with which to generate blocking antibodies to this receptor, while avoiding the dangers of including T cell auto-epitopes.
- Published
- 2008
- Full Text
- View/download PDF
47. Oxidation of ovarian epithelial cancer cells by hypochlorous acid enhances immunogenicity and stimulates T cells that recognize autologous primary tumor.
- Author
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Chiang CL, Ledermann JA, Aitkens E, Benjamin E, Katz DR, and Chain BM
- Subjects
- Aged, CD40 Antigens biosynthesis, Dendritic Cells cytology, Dendritic Cells metabolism, Female, HLA-A2 Antigen metabolism, Humans, Leukocytes, Mononuclear cytology, Middle Aged, Models, Biological, Oxidants pharmacology, Oxygen chemistry, CD8-Positive T-Lymphocytes metabolism, Hypochlorous Acid pharmacology, Neoplasms, Glandular and Epithelial metabolism, Ovarian Neoplasms metabolism, Oxygen metabolism
- Abstract
Purpose: Hypochlorous acid, a product of neutrophil myeloperoxidase, is a powerful enhancer of antigen processing and presentation. In this study, we examine whether ovarian epithelial cells (SK-OV-3) exposed to hypochlorous acid can stimulate T cells from patients with ovarian epithelial cancer that recognize common tumor antigens as well as autologous tumor., Experimental Design: T cells from human leukocyte antigen (HLA)-A2(+) and HLA-A2(-) patients or healthy controls were stimulated with autologous dendritic cells cocultured with the generic ovarian tumor line SK-OV-3, previously exposed to hypochlorous acid., Results: Hypochlorous acid-treated SK-OV-3 cells drove expansion of CD8(+) T cells from HLA-A2(+) individuals, which recognized the HLA-A2-restricted tumor antigen epitopes of HER-2/neu (E75 and GP2) and MUC1 (M1.1 and M1.2). Up to 4.1% of the T cells were positive for the HER-2/neu KIFGSLAFL epitope using pentamer staining. Dendritic cells loaded with oxidized SK-OV-3 cells and further matured with CD40 agonistic antibody or monophosphoryl lipid A additionally induced CD4(+) class II-restricted responses. Critically, T cells stimulated with mature oxidized SK-OV-3 (but not a control oxidized melanoma cell line) directly recognized autologous tumor cells isolated from patient ascites., Conclusions: Immunization with mature dendritic cells loaded with a generic oxidized tumor cell line stimulates a polyclonal antitumor response that recognizes autologous tumor. These findings suggest a new immunotherapeutic strategy to extend remission in ovarian cancer.
- Published
- 2008
- Full Text
- View/download PDF
48. Glycoprotein-dependent and TLR2-independent innate immune recognition of herpes simplex virus-1 by dendritic cells.
- Author
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Reske A, Pollara G, Krummenacher C, Katz DR, and Chain BM
- Subjects
- Animals, COS Cells, Cell Fusion, Cell Line, Cells, Cultured, Chlorocebus aethiops, Dendritic Cells metabolism, Dendritic Cells virology, Herpesvirus 1, Human metabolism, Humans, Interferon Type I immunology, Interferon Type I metabolism, Interleukin-10 immunology, Interleukin-10 metabolism, Toll-Like Receptor 2 immunology, Toll-Like Receptor 2 metabolism, Viral Envelope Proteins metabolism, Dendritic Cells immunology, Herpesvirus 1, Human immunology, Viral Envelope Proteins immunology
- Abstract
Innate immune recognition is an important early event in the host response to herpes simplex virus-1 (HSV-1) infection. Dendritic cells (DC) play an important sentinel role in this recognition. Previous studies have shown that monocyte-derived DC (MDDC) respond to HSV-1 by up-regulation of costimulatory molecules and type I IFN release, but the molecular targets on the virus recognized by the DC have not been defined. In this study we show that MDDC recognize and respond to the four essential viral glycoproteins, gB, gD, and gHgL, independent of other viral proteins or nucleic acids. DC recognition of these four glycoproteins leads to the up-regulation of CD40, CD83, CD86, and HLA-DR and to the production of IFN-alpha and IL-10, but not IL-12p70. Glutaraldehyde-fixation and nonfunctional gH mutants were used to show that recognition of glycoproteins does not require membrane fusion. The nature of the recognition event was probed further by transfecting glycoproteins individually or in combination, by blocking individual proteins with Abs, or by using mutant gD constructs unable to bind to their known cognate receptors. Unexpectedly, MDDC responses were found to require expression of all four glycoproteins. Furthermore, gD mutants that cannot bind nectin-1 and/or herpesvirus entry mediator can still induce DC maturation. Finally, although HSV-1 can signal via the TLR2 receptor, this receptor does not mediate recognition of glycoproteins. Thus, the complex of the four essential HSV-1 entry glycoproteins on the cell surface can provide a target for innate immune recognition of this virus.
- Published
- 2008
- Full Text
- View/download PDF
49. Quantitative imaging assay for NF-kappaB nuclear translocation in primary human macrophages.
- Author
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Noursadeghi M, Tsang J, Haustein T, Miller RF, Chain BM, and Katz DR
- Subjects
- Active Transport, Cell Nucleus, Cell Culture Techniques, Cell Line, Cell Nucleus drug effects, Cells, Cultured, Dose-Response Relationship, Drug, Genes, Reporter, Humans, Lipopeptides, Lipopolysaccharides pharmacology, Macrophages drug effects, Peptides pharmacology, Polymyxin B pharmacology, Reproducibility of Results, Signal Processing, Computer-Assisted, Software, Spectrophotometry, Time Factors, Toll-Like Receptor 2 genetics, Toll-Like Receptor 2 metabolism, Transcription Factor RelA genetics, Transfection, Cell Nucleus metabolism, Macrophage Activation drug effects, Macrophages metabolism, Microscopy, Confocal, Microscopy, Fluorescence, Transcription Factor RelA metabolism
- Abstract
Quantitative measurement of NF-kappaB nuclear translocation is an important research tool in cellular immunology. Established methodologies have a number of limitations, such as poor sensitivity, high cost or dependence on cell lines. Novel imaging methods to measure nuclear translocation of transcriptionally active components of NF-kappaB are being used but are also partly limited by the need for specialist imaging equipment or image analysis software. Herein we present a method for quantitative detection of NF-kappaB rel A nuclear translocation, using immunofluorescence microscopy and the public domain image analysis software ImageJ that can be easily adopted for cellular immunology research without the need for specialist image analysis expertise and at low cost. The method presented here is validated by demonstrating the time course and dose response of NF-kappaB nuclear translocation in primary human macrophages stimulated with LPS, and by comparison with a commercial NF-kappaB activation reporter cell line.
- Published
- 2008
- Full Text
- View/download PDF
50. Haematopoietic development and immunological function in the absence of cathepsin D.
- Author
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Tulone C, Uchiyama Y, Novelli M, Grosvenor N, Saftig P, and Chain BM
- Subjects
- Animals, Blotting, Western, Bone Marrow Transplantation, Dendritic Cells immunology, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Fluorescent Antibody Technique, Immunohistochemistry, Inclusion Bodies metabolism, Lysosomes metabolism, Mice, Mice, Inbred BALB C, Microscopy, Confocal, Radiation Chimera, Spleen cytology, Spleen immunology, T-Lymphocytes immunology, Thymus Gland cytology, Thymus Gland immunology, Cathepsin D deficiency, Cathepsin D immunology, Hematopoiesis, Immunity, Innate
- Abstract
Background: Cathepsin D is a well-characterized aspartic protease expressed ubiquitously in lysosomes. Cathepsin D deficiency is associated with a spectrum of pathologies leading ultimately to death. Cathepsin D is expressed at high levels in many cells of the immune system, but its role in immune function is not well understood. This study examines the reconstitution and function of the immune system in the absence of cathepsin D, using bone marrow radiation chimaeras in which all haematopoietic cells are derived from cathepsin D deficient mice., Results: Cathepsin D deficient bone marrow cells fully reconstitute the major cellular components of both the adaptive and innate immune systems. Spleen cells from cathepsin D deficient chimaeric mice contained an increased number of autofluorescent granules characteristic of lipofuscin positive lysosomal storage diseases. Biochemical and ultrastructural changes in cathepsin D deficient spleen are consistent with increased autolysosomal activity. Chimaeric mice were immunised with either soluble (dinitrophenylated bovine gamma globulin) or particulate (sheep red blood cells) antigens. Both antigens induced equivalent immune responses in wild type or cathepsin D deficient chimaeras., Conclusion: All the parameters of haematopoietic reconstitution and adaptive immunity which were measured in this study were found to be normal in the absence of cathepsin D, even though cathepsin D deficiency leads to dysregulation of lysosomal function.
- Published
- 2007
- Full Text
- View/download PDF
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