register interest

Professor Ling-Pei Ho

Research Area: Immunology
Technology Exchange: Cellular immunology
Scientific Themes: Immunology & Infectious Disease
Keywords: Lungs, Immuneregulation, Natural Killer T (iNKT) cells, Monocytes, Lung Fibrosis and Influenza virus infection
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We are interested in how immunological responses impact on mechanisms of lung injury and repair. The projects are divided into mechanistic and translational studies. The mechanistic studies question how innate immune cells like iNKT cells, MDSCs and monocyte-macrophage lineage impact on outcome in severe influenza virus infection and progression of lung fibrosis. These studies inform, drive and allow us to test mechanistic hypotheses in/from our human work.

In the translational space, our focus is on new or improved therapy for lung fibrosis. Our diseases of interest are idiopathic pulmonary fibrosis (IPF) and fibrotic sarcoidosis, and we target the interface between cellular immunology, disease mechanisms and early clinical trials.

Current projects in the group

  • Role of epithelial CD1d in local lung immune homeostasis and responses
  • Impact of monocyte subtypes, monocyte-derived macrophages and MDSCs on immune response to pathogens
  • Development of human lung organoid models for idiopathic pulmonary fibrosis
  • Contribution of monocyte-macrophage subtypes to acceleration and acute exacerbations of idiopathic pulmonary fibrosis 
  • Examination of M-CSF/CSF-1R pathway as a potential therapy for IPF

Name Department Institution Country
Dr Luzheng Xue NDM Research Building Oxford University, NDM Research Building United Kingdom
Prof Tao Dong (RDM) Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Dr Fergus Gleeson Experimental Medicine Division University of Oxford United Kingdom
Elderfield RA, Watson SJ, Godlee A, Adamson WE, Thompson CI, Dunning J, Fernandez-Alonso M, Blumenkrantz D et al. 2014. Accumulation of Human-Adapting Mutations during Circulation of A(H1N1)pdm09 Influenza Virus in Humans in the United Kingdom Journal of Virology, 88 (22), pp. 13269-13283. | Read more

Crawshaw A, Kendrick YR, McMichael AJ, Ho LP. 2014. Abnormalities in iNKT cells are associated with impaired ability of monocytes to produce IL-10 and suppress T-cell proliferation in sarcoidosis. Eur J Immunol, 44 (7), pp. 2165-2174. | Show Abstract | Read more

Sarcoidosis is a multisystem granulomatous disorder characterized by marked T-cell expansion of T helper 1 (Th1) cells. The cause of T-cell overactivity is unknown. We hypothesized that interleukin-10 (IL-10) production by a yet undefined cell type might be defective, resulting in loss of regulation of T-cell activity. Focusing on IL-10-producing monocytes, we first showed that monocytes isolated from the peripheral blood of corticosteroid-naïve sarcoidosis patients (n = 51) produced less IL-10 compared to controls, and were less able to suppress T-cell proliferation. In addition, monocytic IL-10 production correlated negatively with disease activity score. As invariant natural killer T (iNKT) cells are known to both interact with monocytes and be reduced in sarcoidosis patients, we then asked whether iNKT-specific defects might be responsible for this reduced IL-10 production. We found that greater numbers of circulating iNKT cells was associated with higher IL-10 production. Moreover, iNKT cells enhanced monocytic IL-10 production in vitro. Defective IL-10 production and T-cell suppression by sarcoidosis monocytes could be restored following their coculture with iNKT cells, in a CD1d- and cell contact-dependent process. We suggest that reduced iNKT-cell numbers in sarcoidosis may lead to impaired monocytic IL-10 production and unchecked T-cell expansion in sarcoidosis. These findings provide fresh insight into the mechanism of sarcoidosis disease, and interaction between iNKT cells and monocytes.

Cole SL, Benam KH, McMichael AJ, Ho LP. 2014. Involvement of the 4-1BB/4-1BBL pathway in control of monocyte numbers by invariant NKT cells. J Immunol, 192 (8), pp. 3898-3907. | Show Abstract | Read more

4-1BB is expressed on invariant (i)NKT cells, but its role is unclear. We showed previously that iNKT cells are involved in control of monocyte numbers during influenza A virus (IAV) infection and now question the role of the 4-1BB costimulatory pathway in the cross-talk between these cells. We found that iNKT cells and monocytes interact to promote expression of 4-1BB and 4-1BBL, respectively. Blockade of 4-1BB/L pathway under resting coculture conditions increased apoptosis of iNKT cells and monocytes. However, activation of iNKT cells overrides this survival signal, causing marked apoptosis of monocytes independent of 4-1BB/L. Blocking 4-1BBL in alpha-galactosylceramide-activated iNKT-monocyte cocultures reduced iNKT proliferation and abrogated monocytic IL-12 production. In vivo, expression of 4-1BB and 4-1BBL is increased on iNKT cells and Ly6C(hi) monocytes, respectively, during IAV infection, and there were lower frequencies of apoptosing Ly6C(hi) monocytes in the blood of iNKT knockout mice and higher numbers of monocytes in lungs compared with infected wild-type mice. Adoptive transfer of iNKT cells into the lungs of these mice reduced lung Ly6C(hi) monocytes levels, even when iNKT cells were preincubated with 4-1BB blocking Abs. These findings suggest that under resting conditions, 4-1BB/L engagement during iNKT-monocyte interaction promotes survival of these cells. When iNKT cells are activated, whether by alpha-galactosylceramide or during IAV infection, iNKT cells induced apoptosis of monocytes via a 4-1BB/L-independent mechanism, reducing monocyte numbers. 4-1BB/L costimulation amplified monocyte-mediated proliferation of iNKT cells, indirectly providing a method for monocytes to control their own numbers during infection.

Knox R, Nettleship JE, Chang VT, Hui ZK, Santos AM, Rahman N, Ho LP, Owens RJ, Davis SJ. 2013. A streamlined implementation of the glutamine synthetase-based protein expression system. BMC Biotechnol, 13 (1), pp. 74. | Show Abstract | Read more

BACKGROUND: The glutamine synthetase-based protein expression system is widely used in industry and academia for producing recombinant proteins but relies on the cloning of transfected cells, necessitating substantial investments in time and handling. We streamlined the production of protein-producing cultures of Chinese hamster ovary cells using this system by co-expressing green fluorescent protein from an internal ribosomal entry site and selecting for high green fluorescent protein-expressing cells using fluorescence-activated cell sorting. RESULTS: Whereas other expression systems utilizing green fluorescent protein and fluorescence-activated cell sorting-based selection have relied on two or more sorting steps, we obtained stable expression of a test protein at levels >50% of that of an "average" clone and ~40% that of the "best" clone following a single sorting step. Versus clone-based selection, the principal savings are in the number of handling steps (reduced by a third), handling time (reduced by 70%), and the time needed to produce protein-expressing cultures (reduced by ~3 weeks). Coupling the glutamine synthetase-based expression system with product-independent selection in this way also facilitated the production of a hard-to-assay protein. CONCLUSION: Utilizing just a single fluorescence-activated cell sorting-based selection step, the new streamlined implementation of the glutamine synthetase-based protein expression system offers protein yields sufficient for most research purposes, where <10 mg/L of protein expression is often required but relatively large numbers of constructs frequently need to be trialed.

Bloom CI, Graham CM, Berry MP, Rozakeas F, Redford PS, Wang Y, Xu Z, Wilkinson KA et al. 2013. Transcriptional blood signatures distinguish pulmonary tuberculosis, pulmonary sarcoidosis, pneumonias and lung cancers. PLoS One, 8 (8), pp. e70630. | Show Abstract | Read more

RATIONALE: New approaches to define factors underlying the immunopathogenesis of pulmonary diseases including sarcoidosis and tuberculosis are needed to develop new treatments and biomarkers. Comparing the blood transcriptional response of tuberculosis to other similar pulmonary diseases will advance knowledge of disease pathways and help distinguish diseases with similar clinical presentations. OBJECTIVES: To determine the factors underlying the immunopathogenesis of the granulomatous diseases, sarcoidosis and tuberculosis, by comparing the blood transcriptional responses in these and other pulmonary diseases. METHODS: We compared whole blood genome-wide transcriptional profiles in pulmonary sarcoidosis, pulmonary tuberculosis, to community acquired pneumonia and primary lung cancer and healthy controls, before and after treatment, and in purified leucocyte populations. MEASUREMENTS AND MAIN RESULTS: An Interferon-inducible neutrophil-driven blood transcriptional signature was present in both sarcoidosis and tuberculosis, with a higher abundance and expression in tuberculosis. Heterogeneity of the sarcoidosis signature correlated significantly with disease activity. Transcriptional profiles in pneumonia and lung cancer revealed an over-abundance of inflammatory transcripts. After successful treatment the transcriptional activity in tuberculosis and pneumonia patients was significantly reduced. However the glucocorticoid-responsive sarcoidosis patients showed a significant increase in transcriptional activity. 144-blood transcripts were able to distinguish tuberculosis from other lung diseases and controls. CONCLUSIONS: Tuberculosis and sarcoidosis revealed similar blood transcriptional profiles, dominated by interferon-inducible transcripts, while pneumonia and lung cancer showed distinct signatures, dominated by inflammatory genes. There were also significant differences between tuberculosis and sarcoidosis in the degree of their transcriptional activity, the heterogeneity of their profiles and their transcriptional response to treatment.

Zhang YH, Zhao Y, Li N, Peng YC, Giannoulatou E, Jin RH, Yan HP, Wu H et al. 2013. Interferon-induced transmembrane protein-3 genetic variant rs12252-C is associated with severe influenza in Chinese individuals. Nat Commun, 4 pp. 1418. | Show Abstract | Read more

The SNP rs12252-C allele alters the function of interferon-induced transmembrane protein-3 increasing the disease severity of influenza virus infection in Caucasians, but the allele is rare. However, rs12252-C is much more common in Han Chinese. Here we report that the CC genotype is found in 69% of Chinese patients with severe pandemic influenza A H1N1/09 virus infection compared with 25% in those with mild infection. Specifically, the CC genotype was estimated to confer a sixfold greater risk for severe infection than the CT and TT genotypes. More importantly, because the risk genotype occurs with such a high frequency, its effect translates to a large population-attributable risk of 54.3% for severe infection in the Chinese population studied compared with 5.4% in Northern Europeans. Interferon-induced transmembrane protein-3 genetic variants could, therefore, have a strong effect of the epidemiology of influenza in China and in people of Chinese descent.

Martinez FO, Helming L, Milde R, Varin A, Melgert BN, Draijer C, Thomas B, Fabbri M et al. 2013. Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences. Blood, 121 (9), pp. e57-e69. | Show Abstract | Read more

The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4–activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Although mouse models are widely used for macrophage research, translation to the human can be problematic and the human macrophage system remains poorly described. In the present study, we analyzed and compared the transcriptome and proteome of human and murine macrophages under resting conditions (M0) and after IL-4 activation (M2). We provide a resource for tools enabling macrophage detection in human tissues by identifying a set of 87 macrophage-related genes. Furthermore, we extend current understanding of M2 activation in different species and identify Transglutaminase 2 as a conserved M2 marker that is highly expressed by human macrophages and monocytes in the prototypic Th2 pathology asthma.

Zhao Y, Zhang YH, Denney L, Young D, Powell TJ, Peng YC, Li N, Yan HP et al. 2012. High levels of virus-specific CD4+ T cells predict severe pandemic influenza A virus infection. Am J Respir Crit Care Med, 186 (12), pp. 1292-1297. | Show Abstract | Read more

RATIONALE: T-cell responses have been implicated in control and exacerbation of lung injury during influenza A virus (IAV) infection. OBJECTIVES: To examine the breadth and magnitude of influenza-specific CD4(+) and CD8(+) T-cell responses during acute phase of infection. METHODS: Influenza-specific T-cell response to the entire pandemic H1N1/09 IAV proteome and T cell-related cytokine levels were measured in blood from previously healthy individuals with mild (n = 32) and severe (n = 16) IAV infection during the 2009 influenza pandemic. Virus-specific T-cell response in lung and blood was also performed in two acutely infected, severely ill patients using fluorescent-conjugated pdmH1N1/09 Matrix-MHC-I tetrameric complexes. MEASUREMENTS AND MAIN RESULTS: Strong and broad CD4(+) but not CD8(+) T-cell responses were observed in the blood, and were higher in those with severe disease. Antigen-specific CD8(+) T cells in the lungs were on average 45-fold higher compared with blood in severely ill patients. Paradoxically, in patients with severe disease, IL-17, IL-2, IL-4, and IFN-γ levels were significantly decreased. CONCLUSIONS: High levels of circulating virus-specific CD4(+) T cells to two viral internal proteins (nucleoprotein and matrix) in the first phase of infection are associated with subsequent development of severe IAV infection. This finding could be an early and specific marker for ensuing clinical deterioration. Contrasting levels of antigen-specific CD8(+) T cells in lungs and blood have implications on design and analysis of clinical trials for T-cell vaccines because measurements of T cells in the periphery may not reflect events in the lungs.

Wegmann F, Gartlan KH, Harandi AM, Brinckmann SA, Coccia M, Hillson WR, Kok WL, Cole S et al. 2012. Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens. Nat Biotechnol, 30 (9), pp. 883-888. | Show Abstract | Read more

Protection against mucosally transmitted infections probably requires immunity at the site of pathogen entry, yet there are no mucosal adjuvant formulations licensed for human use. Polyethyleneimine (PEI) represents a family of organic polycations used as nucleic acid transfection reagents in vitro and DNA vaccine delivery vehicles in vivo. Here we show that diverse PEI forms have potent mucosal adjuvant activity for viral subunit glycoprotein antigens. A single intranasal administration of influenza hemagglutinin or herpes simplex virus type-2 (HSV-2) glycoprotein D with PEI elicited robust antibody-mediated protection from an otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen, which were taken up by antigen-presenting cells in vitro and in vivo, promoted dendritic cell trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host double-stranded DNA that triggered Irf3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use.

Denney L, Kok WL, Cole SL, Sanderson S, McMichael AJ, Ho LP. 2012. Activation of invariant NKT cells in early phase of experimental autoimmune encephalomyelitis results in differentiation of Ly6Chi inflammatory monocyte to M2 macrophages and improved outcome. J Immunol, 189 (2), pp. 551-557. | Show Abstract | Read more

Neuropathology in multiple sclerosis is closely linked to presence of macrophages in the CNS. Both M1 (inflammatory) and M2 (alternatively activated, noninflammatory) macrophages are found in the inflamed CNS and thought to differentiate from infiltrating monocytes. It is unclear whether the balance of M1 and M2 macrophages can be altered and whether this affects disease outcome. We show in this article that Ly6C(hi) inflammatory monocytes are the early and dominant infiltrating cells in the CNS during experimental autoimmune encephalomyelitis, a model for the acute phase of multiple sclerosis. Activation of invariant NKT (iNKT) cells reduced the frequency of Ly6C(hi) monocytes and increased the proportion of M2 macrophages in the CNS with associated improvement in neurologic impairment. In contrast, iNKT-deficient mice showed higher numbers of Ly6C(hi) monocytes, reduced M2, and much more severe disease. Adoptive transfer of M2-enriched cells to iNKT-deficient mice markedly improved neurologic impairment. In vitro and in vivo experiments showed that iNKT cells promote differentiation of monocytes to M2 macrophages in an IL-4 and CD1d-dependent process. These findings indicate that infiltrating Ly6C(hi) inflammatory monocytes are early players in acute neuroinflammation and that their frequency and differentiation can be influenced by activation of iNKT cells with resultant improvement in disease outcome.

Everitt AR, Clare S, Pertel T, John SP, Wash RS, Smith SE, Chin CR, Feeley EM et al. 2012. IFITM3 restricts the morbidity and mortality associated with influenza Nature,

Kok WL, Denney L, Benam K, Cole S, Clelland C, McMichael AJ, Ho LP. 2012. Pivotal Advance: Invariant NKT cells reduce accumulation of inflammatory monocytes in the lungs and decrease immune-pathology during severe influenza A virus infection. J Leukoc Biol, 91 (3), pp. 357-368. | Show Abstract | Read more

Little is known of how a strong immune response in the lungs is regulated to minimize tissue injury during severe influenza A virus (IAV) infection. Here, using a model of lethal, high-pathogenicity IAV infection, we first show that Ly6C(hi)Ly6G(-) inflammatory monocytes, and not neutrophils, are the main infiltrate in lungs of WT mice. Mice devoid of iNKT cells (Jα18(-/-) mice) have increased levels of inflammatory monocytes, which correlated with increased lung injury and mortality (but not viral load). Activation of iNKT cells correlated with reduction of MCP-1 levels and improved outcome. iNKT cells were able to selectively lyse infected, MCP-1-producing monocytes in vitro, in a CD1d-dependent process. Our study provides a detailed profile and kinetics of innate immune cells in the lungs during severe IAV infection, highlighting inflammatory monocytes as the major infiltrate and identifying a role for iNKT cells in control of these cells and lung immune-pathology.

Armitage AE, Eddowes LA, Gileadi U, Cole S, Spottiswoode N, Selvakumar TA, Ho LP, Townsend AR, Drakesmith H. 2011. Hepcidin regulation by innate immune and infectious stimuli. Blood, 118 (15), pp. 4129-4139. | Show Abstract | Read more

Hepcidin controls the levels and distribution of iron, an element whose availability can influence the outcome of infections. We investigated hepcidin regulation by infection-associated cytokines, pathogen-derived molecules, and whole pathogens in vitro and in vivo. We found that IL-22, an effector cytokine implicated in responses to extracellular infections, caused IL-6-independent hepcidin up-regulation in human hepatoma cells, suggesting it might represent an additional inflammatory hepcidin agonist. Like IL-6, IL-22 caused phosphorylation of STAT3 and synergized with BMP6 potentiating hepcidin induction. In human leukocytes, IL-6 caused potent, transient hepcidin up-regulation that was augmented by TGF-β1. Pathogen-derived TLR agonists also stimulated hepcidin, most notably the TLR5 agonist flagellin in an IL-6-dependent manner. In contrast, leukocyte hepcidin induction by heat-killed Candida albicans hyphae was IL-6-independent, but partially TGF-β-dependent. In a murine acute systemic candidiasis model, C albicans strongly stimulated hepcidin, accompanied by a major reduction in transferrin saturation. Similarly, hepcidin was up-regulated with concomitant lowering of serum iron during acute murine Influenza A/PR/8/34 virus (H1N1) infection. This intracellular pathogen also stimulated hepcidin expression in leukocytes and hepatoma cells. Together, these results indicate that hepcidin induction represents a component of the innate immune response to acute infection, with the potential to affect disease pathogenesis.

Benam KH, Kok WL, McMichael AJ, Ho LP. 2011. Alternative spliced CD1d transcripts in human bronchial epithelial cells PLoS ONE, 6 (8), | Show Abstract | Read more

CD1d is a MHC I like molecule which presents glycolipid to natural killer T (NKT) cells, a group of cells with diverse but critical immune regulatory functions in the immune system. These cells are required for optimal defence against bacterial, viral, protozoan, and fungal infections, and control of immune-pathology and autoimmune diseases. CD1d is expressed on antigen presenting cells but also found on some non-haematopoietic cells. However, it has not been observed on bronchial epithelium, a site of active host defence in the lungs. Here, we identify for the first time, CD1D mRNA variants and CD1d protein expression on human bronchial epithelial cells, describe six alternatively spliced transcripts of this gene in these cells; and show that these variants are specific to epithelial cells. These findings provide the basis for investigations into a role for CD1d in lung mucosal immunity. © 2011 Benam et al.

Benam KH, Kok WL, McMichael AJ, Ho LP. 2011. Alternative spliced CD1d transcripts in human bronchial epithelial cells. PLoS One, 6 (8), pp. e22726. | Show Abstract | Read more

CD1d is a MHC I like molecule which presents glycolipid to natural killer T (NKT) cells, a group of cells with diverse but critical immune regulatory functions in the immune system. These cells are required for optimal defence against bacterial, viral, protozoan, and fungal infections, and control of immune-pathology and autoimmune diseases. CD1d is expressed on antigen presenting cells but also found on some non-haematopoietic cells. However, it has not been observed on bronchial epithelium, a site of active host defence in the lungs. Here, we identify for the first time, CD1D mRNA variants and CD1d protein expression on human bronchial epithelial cells, describe six alternatively spliced transcripts of this gene in these cells; and show that these variants are specific to epithelial cells. These findings provide the basis for investigations into a role for CD1d in lung mucosal immunity.

Ho LP. 2010. Exosomes in lungs of patients with sarcoidosis: a contributor to immune pathogenesis or just another by-product of heightened immune activity? Thorax, 65 (11), pp. 947-948. | Read more

Crawshaw AP, Wotton CJ, Yeates DG, Goldacre MJ, Ho LP. 2011. Evidence for association between sarcoidosis and pulmonary embolism from 35-year record linkage study. Thorax, 66 (5), pp. 447-448. | Read more

Lockstone HE, Sanderson S, Kulakova N, Baban D, Leonard A, Kok WL, McGowan S, McMichael AJ, Ho LP. 2010. Gene set analysis of lung samples provides insight into pathogenesis of progressive, fibrotic pulmonary sarcoidosis. Am J Respir Crit Care Med, 181 (12), pp. 1367-1375. | Show Abstract | Read more

RATIONALE: Approximately 60 to 70% of patients with pulmonary sarcoidosis have disease that resolves spontaneously; the rest follow a chronic course with varying levels of fibrosis. It is unclear why some patients progress and if treatment affects outcome. OBJECTIVES: To determine differential gene expression profile in lungs of patients with self-limiting sarcoidosis compared to those with progressive-fibrotic disease, and to analyze the biological relevance of these differentially expressed genes. METHODS: We examined microarray expression of 26,626 genes in transbronchial biopsies of granulomatous areas in lungs of patients with active but self-limiting (n = 8) versus those with active, progressive (+/- fibrotic) pulmonary disease (n = 7). MEASUREMENTS AND MAIN RESULTS: Three hundred thirty-four genes were differentially expressed between the two groups (P < 0.01, Bayesian moderated t test). Gene Set Enrichment Analysis showed over-representation of gene-sets (defined by Gene Ontology) related to host immune activation, proliferation, and defense, among genes up-regulated in the progressive-fibrotic group (FDR q < 0.0001 for the top 43 gene sets), and a marked enrichment of, and similarity in gene expression profiles between, progressive-fibrotic sarcoidosis and hypersensitivity pneumonitis (HP), (q < 0.001), but not idiopathic pulmonary fibrosis (IPF). CONCLUSIONS: The findings suggest that patients with progressive/fibrotic pulmonary sarcoidosis have intense immune activity related to host defense in their lungs, with processes more similar to HP than IPF. The study also demonstrates that transbronchial lung biopsy samples can provide good-quality RNA for gene expression profiling, supporting its potential use as a prognostic classifier for pulmonary sarcoidosis.

Krashias G, Simon AK, Wegmann F, Kok WL, Ho LP, Stevens D, Skehel J, Heeney JL, Moghaddam AE, Sattentau QJ. 2010. Potent adaptive immune responses induced against HIV-1 gp140 and influenza virus HA by a polyanionic carbomer. Vaccine, 28 (13), pp. 2482-2489. | Show Abstract | Read more

Carbopol is a polyanionic carbomer gel used in man for a variety of topical applications and drug delivery purposes. Here we show that subcutaneous administration of carbopol with glycoprotein antigens elicits unusually strong specific adaptive immune responses in mice. Recombinant soluble HIV-1 envelope glycoprotein (Env)-based antigen formulated in carbopol was at least as potent at stimulating Env-specific B and T cell responses as Freund's Complete Adjuvant, and significantly more potent than aluminium salts. The antigen-specific T cell immune response elicited both Th1 and Th2 cytokines including high titers of IFN-gamma, IL-2 and IL-4, and drove a Th1 isotype-switched antibody response. Mice immunized with a low dose of purified influenza HA in carbopol generated high titers of anti-HA antibodies and were protected from lethal challenge and disease with live virus. Similarly, immunization of mice with the melanoma cell line B16F10 formulated in carbopol significantly delayed tumor growth. We propose that carbopol, or related cross-linked polyacrylic acid analogues, may have promise for use as systemic vaccine adjuvants in man.

Denney L, Aitken C, Li CK, Wilson-Davies E, Kok WL, Clelland C, Rooney K, Young D et al. 2010. Reduction of natural killer but not effector CD8 T lymphocytes in three consecutive cases of severe/lethal H1N1/09 influenza A virus infection. PLoS One, 5 (5), pp. e10675. | Show Abstract | Read more

BACKGROUND: The cause of severe disease in some patients infected with pandemic influenza A virus is unclear. METHODOLOGY/PRINCIPAL FINDINGS: We present the cellular immunology profile in the blood, and detailed clinical (and post-mortem) findings of three patients with rapidly progressive infection, including a pregnant patient who died. The striking finding is of reduction in natural killer (NK) cells but preservation of activated effector CD8 T lymphocytes; with viraemia in the patient who had no NK cells. Comparison with control groups suggests that the reduction of NK cells is unique to these severely ill patients. CONCLUSION/SIGNIFICANCE: Our report shows markedly reduced NK cells in the three patients that we sampled and raises the hypothesis that NK may have a more significant role than T lymphocytes in controlling viral burden when the host is confronted with a new influenza A virus subtype.

Cited:

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Scopus

Denney L, Aitken C, Li CKF, Wilson-Davies E, Kok WL, Clelland C, Rooney K, Young D et al. 2010. Reduction of natural killer but not effector CD8 t lymphoyctes in three consecutive cases of severe/lethal H1N1/09 influenza a virus infection PLoS ONE, 5 (5), | Show Abstract | Read more

Background: The cause of severe disease in some patients infected with pandemic influenza A virus is unclear. Methodology/Principal Findings: We present the cellular immunology profile in the blood, and detailed clinical (and postmortem) findings of three patients with rapidly progressive infection, including a pregnant patient who died. The striking finding is of reduction in natural killer (NK) cells but preservation of activated effector CD8 T lymphocytes; with viraemia in the patient who had no NK cells. Comparison with control groups suggests that the reduction of NK cells is unique to these severely ill patients. Conclusion/Significance: Our report shows markedly reduced NK cells in the three patients that we sampled and raises the hypothesis that NK may have a more significant role than T lymphocytes in controlling viral burden when the host is confronted with a new influenza A virus subtype. © 2010 Denney et al.

Ralston SH, Ho L-P, Helfrich MH, Grabowski PS, Johnston PW, Benjamin N. 1995. Nitric oxide: A cytokine-induced regulator of bone resorption Journal of Bone and Mineral Research, 10 (7), pp. 1040-1049. | Read more

Kulakova N, Urban B, McMichael AJ, Ho LP. 2010. Functional analysis of dendritic cell-T cell interaction in sarcoidosis. Clin Exp Immunol, 159 (1), pp. 82-86. | Show Abstract | Read more

The primary cause of the intense immune response in sarcoidosis is unclear. Potentially, a functional abnormality in dendritic cells (DCs) could cause a reduction in clearance of antigen and downstream persistence in immune activity. In this study, we investigate the interaction between monocyte-derived dendritic cells and T cells in patients with sarcoidosis compared to normal controls (n = 8 each) by examining the kinetics of autologous and allogeneic mixed leucocyte reactions over 9-10 days. We found markedly depressed proliferation kinetics in autologous DC-peripheral blood mononuclear cell (PBMC) co-cultures from sarcoid patients compared to normal subjects. In allogeneic experiments PBMCs from patients showed a reduced response to allogeneic DCs from a single donor, but no difference was observed in the ability of patients and control DCs to stimulate proliferation of allogeneic PBMC from a single donor. We conclude that there is a markedly impaired autologous mixed leucocyte reaction (MLR) in sarcoidosis patients. In allogeneic MLR, monocyte-derived DCs in sarcoidosis were able to stimulate T cells normally, but PBMCs responses were reduced. This contradicts recent published studies on ex vivo isolated myeloid DCs from sarcoidosis patients although, potentially, an in vivo conditioning factor, which reduces DC function in sarcoidosis, could be a unifying explanation for the contrasting findings.

Ho LP, Denney L, Luhn K, Teoh D, Clelland C, McMichael AJ. 2008. Activation of invariant NKT cells enhances the innate immune response and improves the disease course in influenza A virus infection. Eur J Immunol, 38 (7), pp. 1913-1922. | Show Abstract | Read more

Invariant NKT (iNKT) cells have an indubitable role in antiviral immunity, although the mechanisms by which these cells exert their functions are not fully elucidated. With the emerging importance of high-pathogenicity influenza A virus infections in humans, we questioned whether iNKT cells contribute to immune defence against influenza A virus and whether activation of these cells influences outcome. We show that activation of iNKT cells with alpha-galactosylceramide (alpha-GC) during influenza virus infection transiently enhanced early innate immune response without affecting T cell immunity, and reduced early viral titres in lungs of C57BL/6 mice. This is accompanied by a better disease course with improved weight loss profile. Temporal changes in iNKT cells in the liver, blood and lungs suggest activation and migration of iNKT cells from the liver to the lungs in mice that were administered alpha-GC. Improvement in viral titres appears dependent on activation of iNKT cells via the intraperitoneal route since intranasal administration of alpha-GC did not have the same effect. We conclude that activation of iNKT cells enhances early innate immune response in the lungs and contribute to antiviral immunity and improved disease course in influenza A virus infection.

Ho LP. 2007. Natural killer T cells in asthma--toward increased understanding. N Engl J Med, 356 (14), pp. 1466-1468. | Read more

Prasad A, Langford B, Stradling JR, Ho LP. 2006. Exhaled nitric oxide as a screening tool for asthma in school children. Respir Med, 100 (1), pp. 167-173. | Show Abstract | Read more

It is now widely accepted that augmented levels of fractional exhaled nitric oxide (FeNO) reflect airway inflammation and the methodology has been optimised for potential clinical use. We were interested in investigating whether this measurement can be used as a tool to screen and identify school children with asthma. To do this, FeNO was measured using an on-line single exhalation analyser in 368 children aged 8-10 years in six Oxfordshire primary schools, by two investigators blinded to the disease status of the children. The children were then categorised into 'normal', 'atopic asthma', 'non-atopic asthma' and 'atopy only' groups, according to their responses to the ISAAC questionnaire and perusal of the children's medical records kept by their family practitioners. Increased levels of FeNO were found in 'atopic asthmatic', 'non-atopic asthmatics' and 'atopic only' groups (median values of 24.4, 7.8 and 15.3 ppb, respectively, compared to normal controls' of 6.9 ppb). Levels were increased in atopic children regardless of whether they had asthma and were significantly higher than non-atopic asthmatics. We conclude that FeNO measurement is not a useful tool for identifying children with asthma in the community, as increased levels did not discriminate between those with asthmatic and atopic symptoms.

Horváth I, Hunt J, Barnes PJ. 2005. Exhaled breath condensate: methodological recommendations and unresolved questions European Respiratory Journal, 26 (3), pp. 523-548. | Read more

Ho LP, Merlin F, Gaber K, Davies RJ, McMichael AJ, Hugot JP. 2005. CARD 15 gene mutations in sarcoidosis. Thorax, 60 (4), pp. 354-355. | Read more

Ho LP, Urban BC, Thickett DR, Davies RJ, McMichael AJ. 2005. Deficiency of a subset of T-cells with immunoregulatory properties in sarcoidosis. Lancet, 365 (9464), pp. 1062-1072. | Show Abstract | Read more

BACKGROUND: Sarcoidosis is a multisystem disorder that predominantly involves the lungs, characterised by a T-helper 1 (Th1) biased CD4-positive T-cell response and granuloma formation, for which the explanation is unknown. A newly identified subset of T-cells with immunoregulatory functions, CD1d-restricted natural-killer T (NKT) cells, has been shown to protect against disorders with increased CD4-positive Th1 responses in animals. We explored whether abnormalities in these cells are implicated in the pathogenesis of sarcoidosis. METHODS: We generated fluorescence-labelled CD1d-tetrameric complexes and used them, with monoclonal antibodies to Valpha24 and Vbeta11 T-cell receptor, to assess the frequency of CD1d-restricted NKT cells in the peripheral blood of 60 patients with histologically proven sarcoidosis (16 with Lofgren's syndrome) and 60 healthy controls. Lung lymphocytes were also analysed in 16 of the patients with sarcoidosis. FINDINGS: CD1d-restricted NKT cells were absent or greatly reduced in peripheral blood from all patients with sarcoidosis, except those with Lofgren's syndrome (median proportion of lymphocytes 0.01% [IQR 0-0.03] vs 0.06% [0.03-0.12] in controls; p=0.0004). The deficiency was found in both acute and resolved disease and was unrelated to systemic corticosteroid therapy. There was no difference in the proportion of CD1d-restricted NKT cells between peripheral blood and lungs in patients, suggesting that the peripheral-blood deficiency is not due to sequestration of these cells in the lungs. The NKT cells were not observed in mediastinal lymph nodes or granulomatous lesions. CD1d expression on antigen-presenting cells of patients was normal, thus the deficiency of CD1d-restricted NKT cells is not explained by abnormal CD1d expression. INTERPRETATION: Loss of immunoregulation by CD1d-restricted NKT cells could explain the amplified and persistent T-cell activity that characterises sarcoidosis. RELEVANCE TO PRACTICE: Our findings give new insight into the pathogenesis of sarcoidosis and draw attention to a potential target for therapeutic modulation in sarcoidosis.

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Cook LJ, Ho LW, Wang L, Terrenoire E, Brayne C, Evans JG, Xuereb J, Cairns NJ et al. 2005. Candidate gene association studies of genes involved in neuronal cholinergic transmission in Alzheimer's disease suggests choline acetyltransferase as a candidate deserving further study American Journal of Medical Genetics Part B: Neuropsychiatric Genetics, 132B (1), pp. 5-8. | Show Abstract | Read more

Consistent deficits in the cholinergic system are evident in the brains of Alzheimer's Disease (AD) patients, including reductions in the activities of acetylcholine, acetylcholinesterase (AChE), and choline acetyltransferase (ChAT), increased butyrylcholinesterase (BChE) activity, and a selective loss of nicotinic acetylcholine receptors (nAChRs). Accordingly, we have analyzed polymorphisms in the genes encoding AChE, ChAT, BChE, and several of the subunit genes from neuronal nAChRs, for genetic associations with late-onset AD. A significant association for disease was detected for a non-coding polymorphism in ChAT (allele χ12 = 12.84, P = 0.0003; genotype χ22 = 11.89, P = 0.0026). Although replication analysis did not confirm the significance of this finding when the replication samples were considered alone (allele χ12 = 1.02, P=0.32; genotype χ22 = 1.101, P = 0.58) the trends were in the correct direction and a significant association remained when the two sample sets were pooled (allele χ12 = 12.37, P = 0.0004; genotype χ22 = 11.61, P = 0.003). Previous studies have reported significant disease associations for both the K-variant of BChE and the coding ChAT rs3810950 polymorphism with AD. Replication analyses of these two loci failed to detect any significant association for disease in our case-control samples. © 2004 Wiley-Liss, Inc.

Ho LP, Urban BC, Jones L, Ogg GS, McMichael AJ. 2004. CD4(-)CD8alphaalpha subset of CD1d-restricted NKT cells controls T cell expansion. J Immunol, 172 (12), pp. 7350-7358. | Show Abstract

Valpha24 invariant (Valpha24i) CD1d-restricted NKT cells are widely regarded to have immune regulatory properties. They are known to have a role in preventing autoimmune diseases and are involved in optimally mounted immune responses to pathogens and tumor cells. We were interested in understanding how these cells provide protection in autoimmune diseases. We first observed, using EBV/MHC I tetrameric complexes, that expansion of Ag-specific cells in human PBMCs was reduced when CD1d-restricted NKT cells were concomitantly activated. This was accompanied by an increase in a CD4(-)CD8alphaalpha(+) subset of Valpha24i NKT cells. To delineate if a specific subset of NKT cells was responsible for this effect, we generated different subsets of human CD4(-) and CD4(+) Valpha24i NKT clones and demonstrate that a CD4(-)CD8alphaalpha(+) subset with highly efficient cytolytic ability was unique among the clones in being able to suppress the proliferation and expansion of activated T cells in vitro. Activated clones were able to kill CD1d-bearing dendritic or target cells. We suggest that one mechanism by which CD1d-restricted NKT cells can exert a regulatory role is by containing the proliferation of activated T cells, possibly through timely lysis of APCs or activated T cells bearing CD1d.

Ho LP, Davis M, Denison A, Wood FT, Greening AP. 2002. Reduced interleukin-18 levels in BAL specimens from patients with asthma compared to patients with sarcoidosis and healthy control subjects. Chest, 121 (5), pp. 1421-1426. | Show Abstract | Read more

STUDY OBJECTIVES: To investigate whether differing airway interleukin (IL)-18 levels may be implicated in the pathogenesis of asthma and sarcoidosis. SETTING: University teaching hospital. PATIENTS AND METHODS: IL-18 levels were measured in BAL fluid and in the supernatant of lipopolysaccharide (LPS)-stimulated alveolar macrophages obtained by BAL from 15 patients with sarcoidosis, 11 patients with asthma, and 13 healthy subjects. We also examined the relationship between IL-18 levels and macrophage and lymphocyte concentrations in BAL fluid. IL-18 was measured using an in-house enzyme-linked immunosorbent assay. RESULTS: IL-18 levels were significantly lower in BAL fluid from patients with asthma (median, 0.0 pg/mL; interquartile range, 0.0 to 0.0 pg/mL) compared to patients with sarcoidosis (median, 222.0 pg/10(6); interquartile range, 110 to 340 pg/mL; p = 0.009, Mann Whitney rank-sum test) and healthy control subjects (median, 162 pg/mL; interquartile range, 38 to 203 pg/mL; p = 0.025, Mann Whitney rank-sum test). Individual analyses comparing IL-18 levels with BAL macrophage counts, and IL-18 with lymphocyte counts in the three groups showed no correlation between these indexes. The mean levels of IL-18 in unstimulated macrophage supernatants were 410 pg/10(6) cells for patients with asthma, 723.4 pg/10(6) cells for patients with sarcoidosis, and 734.8 pg/10(6) cells for healthy control subjects (p > 0.05). Stimulated macrophages from patients with sarcoidosis responded with increasing amounts of IL-18 at lower doses of LPS than macrophages from healthy control subjects or patients with asthma. CONCLUSION: Our findings suggest that inherently low levels of IL-18 may be associated with the pathogenesis of asthmatic airway inflammation.

Ho LP, Wood FT, Robson A, Innes JA, Greening AP. 2000. Atopy influences exhaled nitric oxide levels in adult asthmatics. Chest, 118 (5), pp. 1327-1331. | Show Abstract | Read more

STUDY OBJECTIVE: To examine whether atopy influences exhaled nitric oxide (NO) levels in adults with established asthma. SETTING: Specialist respiratory unit in a university teaching hospital. PATIENTS: Twenty-eight asthmatics (mean FEV(1), 85.7%) receiving short-acting inhaled bronchodilators and a range of inhaled steroids (0 to 4,000 microg/d). INTERVENTIONS: Subjects were studied on two occasions, 5 to 7 days apart, between September and March. MEASUREMENTS AND RESULTS: On the first day, FEV(1), exhaled NO, and histamine challenge were performed. On the second day, exhaled NO, total IgE, and skin-prick testing to six common allergens were conducted. Exhaled NO was measured with the single exhalation method. We found exhaled NO levels to correlate positively with total IgE (r = 0.43, p = 0.02) and number of positive skin-prick tests (p = 0. 002). By contrast, there was no significant correlation between exhaled NO and FEV(1) or the provocative concentration causing a 20% fall in FEV(1). Subanalyses of steroid-treated and steroid-naive patients in this group revealed the same findings. CONCLUSION: Exhaled NO levels in asthmatics correlate more closely with atopy than with bronchial hyperreactivity and lung function.

Ho LP, Wood FT, Robson A, Innes JA, Greening AP. 2000. The current single exhalation method of measuring exhales nitric oxide is affected by airway calibre. Eur Respir J, 15 (6), pp. 1009-1013. | Show Abstract | Read more

The authors have observed that some patients with acute exacerbations of asthma do not have substantially higher levels of exhaled nitric oxide (NO). The study examined whether this could be explained by the effect of airway calibre on exhaled NO. Exhaled NO, height and forced expiratory volume in one second (FEV1) were measured in 12 steroid-naive asthmatics and 17 normal subjects. For comparison, another group of patients with airways disease (34 cystic fibrosis patients) were also studied. In 20 asthmatics (on various doses of inhaled steroids, 0-3,200 microg x day-1), exhaled NO was measured before and after histamine challenge (immediately after reaching the provocative concentration causing a 20% fall in FEV1) and in 12 of these patients, also after nebulized salbutamol to restore FEV1 to baseline. Studies were also conducted to examine possible confounding effects of repeated spirometry (as would occur in histamine challenge) and nebulized salbutamol alone in exhaled NO levels. Exhaled NO was measured using a single exhalation method with a chemiluminescence analyser at a constant flow rate and mouth pressure. There was a significant correlation between FEV1 and exhaled NO in steroid naive asthmatics (r=0.9, p<0.001) and cystic fibrosis patients (r=-0.48, p<0.05) but not in normal subjects (r=-0.13, p=0.61). Exhaled NO decreased significantly after histamine challenge and returned to baseline after bronchodilation by nebulized salbutamol (mean+/-SEM: 23.6+/-3.6 parts per billion (ppb) (prehistamine), 18.2+/-2.7 ppb (posthistamine) and 23.6+/-3.8 ppb (postsalbutamol) p=0.001). Repeated spirometry and nebulized salbutamol did not affect exhaled NO measurements significantly. Exhaled nitric oxide levels appear to be lower in circumstances of smaller airway diameter. Hence, within a subject nitric oxide levels may be artefactually decreased during bronchoconstriction. This may be caused by increased airflow velocity in constricted airways when the exhalation rate is kept constant.

Cunningham S, McColm JR, Pei Ho L, Greening AP, Marshall TG. 2000. Measurement of inflammatory markers in the breath condensate of children with cystic fibrosis European Respiratory Journal, 15 (5), pp. 955-957. | Read more

Gao PS, Kawada H, Kasamatsu T, Mao XQ, Roberts MH, Miyamoto Y, Yoshimura M, Saitoh Y et al. 2000. Variants of NOS1, NOS2, and NOS3 genes in asthmatics. Biochem Biophys Res Commun, 267 (3), pp. 761-763. | Show Abstract | Read more

Nitric oxide (NO) gas concentrations are higher in expired air in asthmatics. NO is synthesized by three isoforms of NO synthase (NOS) encoded by three distinct genes, NOS1, NOS2, and NOS3. Genome-wide searches have identified linkages to asthma on chromosomes 7, 12, and 17 where these three genes are localized. No association study, however, has been reported to date. To test whether variants of NOS1, NOS2, and NOS3 relate to asthma, a genetic association study was conducted in a British population (n = 300). Intragenic microsatellite variants of NOS1 were significantly associated with asthma [odds ratio (OR) = 2.08, 95% CI: 1.20-3.57 (95% CI), P = 0.008 (Pc = 0.048)], but not with IgE levels. Neither NOS2 nor NOS3 variants showed any association with asthma nor IgE levels. These findings suggest that NOS1 variants may be a significant contributor to asthma in a British population.

Böllert FG, Paton JY, Marshall TG, Calvert J, Greening AP, Innes JA. 1999. Recombinant DNase in cystic fibrosis: a protocol for targeted introduction through n-of-1 trials. Scottish Cystic Fibrosis Group. Eur Respir J, 13 (1), pp. 107-113. | Show Abstract | Read more

Nebulized recombinant human deoxyribonuclease (DNase) reduces sputum viscosity and improves lung function in some cystic fibrosis patients, but individual responses are unpredictable. The aim of this study was to investigate how DNase can be targeted to those cystic fibrosis patients who would benefit most. The Scottish Cystic Fibrosis Group agreed on a randomized, double-blind, placebo-controlled n-of-1 assessment protocol. Patients underwent a maximum of three 4-week assessment periods (2 weeks saline, 2 weeks DNase each). Measurements performed at hospital (exercise, oximetry and spirometry) and home (symptom scores) were used to derive a scoring system to discriminate maximally between DNase and placebo effects. The data on 89 4-week assessments in 52 patients were reported. Twenty-four patients have completed the assessment process (12 responders and 12 nonresponders) to date. Forced expiratory volume in one second (FEVI) was the best discriminator of response, rising by >200 mL after DNase in 33 of 89 (37%) assessments compared with 3 of 89 (3%) after saline. N-of-1 trials, while laborious, permitted genuine treatment effects to be quantified within individuals with confidence, permitting appropriate treatment targeting. This provides a model of how other new expensive therapies may be introduced to maximize patient benefit.

Ho LP, Innes JA, Greening AP. 1998. Exhaled nitric oxide is not elevated in the inflammatory airways diseases of cystic fibrosis and bronchiectasis. Eur Respir J, 12 (6), pp. 1290-1294. | Show Abstract | Read more

Airways inflammation has been associated with increased nitric oxide (NO) in the exhaled breath. It was, therefore, questioned whether exhaled NO could act as an indicator of the severity of airways inflammation in the chronic suppurative lung diseases cystic fibrosis (CF) and bronchiectasis. NO levels in a single exhalation were measured using a chemiluminescence analyser. Thirty-six patients with CF and 16 with bronchiectasis were studied and compared with 22 normal subjects and 35 asthmatic patients. All subjects were nonsmokers and all measurements were made when patients were clinically stable. In addition, exhaled NO was measured in 10 CF patients at the time of onset of an acute infective exacerbation and followed for 7 days during the treatment of the exacerbation in eight of the 10 patients. No significant differences were found in NO levels in patients with CF or bronchiectasis compared with normals (median 4.0, 5.5 and 4.4 parts per billion (ppb), respectively), but all were lower than in asthma patients (10.4 ppb). The NO levels in the CF patients at time of exacerbation were not increased and did not change during treatment. These data show that nitric oxide levels in the exhaled breath of patients with chronic suppurative lung diseases, in contrast to asthma, are not elevated, despite the presence of substantial airways inflammation. Possible explanations include poor diffusion of nitric oxide across increased and viscous airway secretions, removal of nitric oxide by reaction with reactive oxygen species in the inflamed environment and failure of upregulation of epithelial inducible nitric oxide synthase in chronic suppurative conditions.

Ho LP, Innes JA, Greening AP. 1998. Nitrite levels in breath condensate of patients with cystic fibrosis is elevated in contrast to exhaled nitric oxide. Thorax, 53 (8), pp. 680-684. | Show Abstract | Read more

BACKGROUND: Nitric oxide (NO) is released by activated macrophages, neutrophils, and stimulated bronchial epithelial cells. Exhaled NO has been shown to be increased in patients with asthma and has been put forward as a marker of airways inflammation. However, we have found that exhaled NO is not raised in patients with cystic fibrosis, even during infective pulmonary exacerbation. One reason for this may be that excess airway secretions may prevent diffusion of gaseous NO into the airway lumen. We hypothesised that exhaled NO may not reflect total NO production in chronically suppurative airways and investigated nitrite as another marker of NO production. METHODS: Breath condensate nitrite concentration and exhaled NO levels were measured in 21 clinically stable patients with cystic fibrosis of mean age 26 years and mean FEV1 57% and 12 healthy normal volunteers of mean age 31 years. Breath condensate was collected with a validated method which excluded saliva and nasal air contamination and nitrite levels were measured using the Griess reaction. Exhaled NO was measured using a sensitive chemiluminescence analyser (LR2000) at an exhalation rate of 250 ml/s. Fourteen patients with cystic fibrosis had circulating plasma leucocyte levels and differential analysis performed on the day of breath collection. RESULTS: Nitrite levels were significantly higher in patients with cystic fibrosis than in normal subjects (median 1.93 microM compared with 0.33 microM). This correlated positively with circulating plasma leucocytes and neutrophils (r = 0.6). In contrast, exhaled NO values were not significantly different from the normal range (median 3.8 ppb vs 4.4 ppb). There was no correlation between breath condensate nitrite and lung function and between breath condensate nitrite and exhaled NO. CONCLUSIONS: Nitrite levels in breath condensate were raised in stable patients with cystic fibrosis in contrast to exhaled NO. This suggests that nitrite levels may be a more useful measure of NO production and possibly airways inflammation in suppurative airways and that exhaled NO may not reflect total NO production.

Porteous DJ, Dorin JR, McLachlan G, Davidson-Smith H, Davidson H, Stevenson BJ, Carothers AD, Wallace WA et al. 1997. Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther, 4 (3), pp. 210-218. | Show Abstract | Read more

In cystic fibrosis (CF), mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in defective transepithelial ion transport, leading to life shortening inflammatory lung disease. Before lung studies, we tested the safety and efficacy of gene delivery to the nasal epithelium of CF patients using pCMV-CFTR-DOTAP cationic liposome complex. A single dose of 400 micrograms pCMV-CFTR:2.4 mg DOTAP was administered in a randomised, double-blinded fashion to the nasal epithelium of eight CF patients, with a further eight receiving buffer only. Patients were monitored for signs and symptoms for 2 weeks before treatment and 4 weeks after treatment. Inflammatory cells were quantified in a nasal biopsy taken 3 days after treatment. There was no evidence for excess nasal inflammation, circulating inflammatory markers or other adverse events ascribable to active treatment. Gene transfer and expression were assayed by the polymerase chain reaction. Transgene DNA was detected in seven of the eight treated patients up to 28 days after treatment and vector derived CFTR mRNA in two of the seven patients at +3 and +7 days. Transepithelial ion transport was assayed before and after treatment by nasal potential difference during drug perfusion and by SPQ fluorescence halide ion conductance. Partial, sustained correction of CFTR-related functional changes toward normal values were detected in two treated patients. The level of gene transfer and functional correction were comparable to those reported previously using adenoviral vectors or another DNA-liposome complex, but here were sustained and uncompromised by false positives. These results justify further studies with pCMV-CFTR-DOTAP aimed at treating CF lung disease.

Ho LP, Samways JM, Porteous DJ, Dorin JR, Carothers A, Greening AP, Innes JA. 1997. Correlation between nasal potential difference measurements, genotype and clinical condition in patients with cystic fibrosis. Eur Respir J, 10 (9), pp. 2018-2022. | Show Abstract | Read more

In cystic fibrosis (CF), the clinical condition of patients correlates poorly with genotype. One possible explanation is that clinical status is influenced by net preserved chloride secretion rather than the CF mutation. We tested the relationships between residual chloride secretion, as measured by nasal potential difference (PD) and the type of mutation (genotypes expressing apical cystic fibrosis transmembrane conductance regulator (CFTR) protein versus those that do not) and clinical status. Twenty two CF patients (mean age 25.7 yrs, 11 females and 11 males, mean forced expiratory volume in one second (FEV1) 53.1% of predicted) with defined genotypes were recruited. Nasal PD was measured using a standard protocol involving the perfusion of the nasal epithelium with a sodium channel blocker (amiloride), followed by a solution of low chloride and finally with isoprenaline. Patients with apical CFTR protein showed higher residual chloride secretion than those without (amiloride to isoprenaline value of 4.59 and 0.56 mV, respectively, p = 0.01). There was no correlation between mutation type and clinical condition. When these patients were recategorized as "high" (> 10 mV amiloride to isoprenaline response) or "low" (10 mV or less) chloride secretors, we found that the former group had a significantly higher FEV1 (67.7 versus 48.3% pred) and a better pulmonary radiological score (4.14 versus 7.07, by Northern scoring system). These results suggest that some cystic fibrosis patients, regardless of genotype, have an ability to secrete chloride when stimulated with chloride secretatagogues, and this is correlated with a better lung function. These results also have implications for the use of potential difference measurements in novel cystic fibrosis transmembrane conductance regulator replacement trials.

Benamore R, Kendrick YR, Repapi E, Helm E, Cole SL, Taylor S, Ho LP. 2016. CTAS: A CT score to quantify disease activity in pulmonary sarcoidosis Thorax, | Show Abstract | Read more

© 2016 BMJ Publishing Group Ltd & British Thoracic Society.Background A major gap in the management of sarcoidosis is the lack of accessible and objective methods to measure disease activity. Since 90% of patients have pulmonary involvement, we explored if a disease activity score based on thoracic CT scans could address this clinical issue. Methods High-resolution CT scans from 100 consecutive patients with sarcoidosis at a regional sarcoidosis service were scored for extent of CT abnormalities known to relate to granuloma or lymphocytic infiltration from published CT-pathological studies. These individual abnormality scores were then correlated against serum ACE, sIL-2R and change in FVC to identify CT abnormalities that reflect contemporaneous disease activity. The sum of these scores, or CT Activity Score (CTAS), was then validated against FVC response to treatment. Findings CT extent scores for nodularity, ground-glass opacification, interlobular septal thickening and consolidation correlated significantly with at least one of the disease activity parameters and were used to form CTAS. CTAS was found to predict FVC response to treatment at 1 year and was highly reproducible between radiologists. An abbreviated CTAS (aCTAS), constructed from presence or absence of the four CT abnormalities, also showed significant correlation with FVC response to treatment. CTAS and aCTAS also correlated with response to treatment in the fibrotic subgroup. Interpretation CTAS provides a concept for an objective and reproducible CT scoring method to quantify disease activity in sarcoidosis. The score can potentially be used to stratify patients according to disease activity, determine response to treatment and establish if fibrotic sarcoidosis is active.

McLachlan G, Ho LP, Davidson-Smith H, Samways J, Davidson H, Stevenson BJ, Carothers AD, Alton EW et al. 1996. Laboratory and clinical studies in support of cystic fibrosis gene therapy using pCMV-CFTR-DOTAP. Gene Ther, 3 (12), pp. 1113-1123. | Show Abstract

The first phase I study of cystic fibrosis gene therapy using cationic liposomes to deliver the cystic fibrosis conductance regulator gene to the nose reported partial and transient correction of the nasal transepithelial ion transport defect, While encouraging, further improvements will be required if this form of treatment is to be of therapeutic value. We tested a new formulation, pCMV-CFTR-DOTAP. The complex is stable for 10 days and effective at correcting the electrophysiological deficit in the trachea of CF mutant mice at 8 or 9 days after intratracheal instillation. Reliable protocols for consistent detection of as few as 10 molecules of CFTR mRNA and DNA in nasal brushing samples are described, Both vector and DNA have been produced to Good Manufacturing Practice standard, Nasal potential difference measurements developed at the National Heart and Lung Institute to assess the CFTR ion channel activity in CF patients replicated well at the Scottish Adult Cystic Fibrosis Service. The SPO fluorescence assay for halide ion conductance in nasal brushings has also been tested. These establish baseline conditions in the Scottish CF cohort from which evidence for correction can be judged under clinical trial conditions. These studies formed the basis for regulatory approval of a randomised, placebo controlled double-blind phase I research study.

Crawshaw A, Kendrick YR, McMichael AJ, Ho LP. 2014. Abnormalities in iNKT cells are associated with impaired ability of monocytes to produce IL-10 and suppress T-cell proliferation in sarcoidosis. Eur J Immunol, 44 (7), pp. 2165-2174. | Show Abstract | Read more

Sarcoidosis is a multisystem granulomatous disorder characterized by marked T-cell expansion of T helper 1 (Th1) cells. The cause of T-cell overactivity is unknown. We hypothesized that interleukin-10 (IL-10) production by a yet undefined cell type might be defective, resulting in loss of regulation of T-cell activity. Focusing on IL-10-producing monocytes, we first showed that monocytes isolated from the peripheral blood of corticosteroid-naïve sarcoidosis patients (n = 51) produced less IL-10 compared to controls, and were less able to suppress T-cell proliferation. In addition, monocytic IL-10 production correlated negatively with disease activity score. As invariant natural killer T (iNKT) cells are known to both interact with monocytes and be reduced in sarcoidosis patients, we then asked whether iNKT-specific defects might be responsible for this reduced IL-10 production. We found that greater numbers of circulating iNKT cells was associated with higher IL-10 production. Moreover, iNKT cells enhanced monocytic IL-10 production in vitro. Defective IL-10 production and T-cell suppression by sarcoidosis monocytes could be restored following their coculture with iNKT cells, in a CD1d- and cell contact-dependent process. We suggest that reduced iNKT-cell numbers in sarcoidosis may lead to impaired monocytic IL-10 production and unchecked T-cell expansion in sarcoidosis. These findings provide fresh insight into the mechanism of sarcoidosis disease, and interaction between iNKT cells and monocytes.

Cole SL, Benam KH, McMichael AJ, Ho LP. 2014. Involvement of the 4-1BB/4-1BBL pathway in control of monocyte numbers by invariant NKT cells. J Immunol, 192 (8), pp. 3898-3907. | Show Abstract | Read more

4-1BB is expressed on invariant (i)NKT cells, but its role is unclear. We showed previously that iNKT cells are involved in control of monocyte numbers during influenza A virus (IAV) infection and now question the role of the 4-1BB costimulatory pathway in the cross-talk between these cells. We found that iNKT cells and monocytes interact to promote expression of 4-1BB and 4-1BBL, respectively. Blockade of 4-1BB/L pathway under resting coculture conditions increased apoptosis of iNKT cells and monocytes. However, activation of iNKT cells overrides this survival signal, causing marked apoptosis of monocytes independent of 4-1BB/L. Blocking 4-1BBL in alpha-galactosylceramide-activated iNKT-monocyte cocultures reduced iNKT proliferation and abrogated monocytic IL-12 production. In vivo, expression of 4-1BB and 4-1BBL is increased on iNKT cells and Ly6C(hi) monocytes, respectively, during IAV infection, and there were lower frequencies of apoptosing Ly6C(hi) monocytes in the blood of iNKT knockout mice and higher numbers of monocytes in lungs compared with infected wild-type mice. Adoptive transfer of iNKT cells into the lungs of these mice reduced lung Ly6C(hi) monocytes levels, even when iNKT cells were preincubated with 4-1BB blocking Abs. These findings suggest that under resting conditions, 4-1BB/L engagement during iNKT-monocyte interaction promotes survival of these cells. When iNKT cells are activated, whether by alpha-galactosylceramide or during IAV infection, iNKT cells induced apoptosis of monocytes via a 4-1BB/L-independent mechanism, reducing monocyte numbers. 4-1BB/L costimulation amplified monocyte-mediated proliferation of iNKT cells, indirectly providing a method for monocytes to control their own numbers during infection.

Bloom CI, Graham CM, Berry MP, Rozakeas F, Redford PS, Wang Y, Xu Z, Wilkinson KA et al. 2013. Transcriptional blood signatures distinguish pulmonary tuberculosis, pulmonary sarcoidosis, pneumonias and lung cancers. PLoS One, 8 (8), pp. e70630. | Show Abstract | Read more

RATIONALE: New approaches to define factors underlying the immunopathogenesis of pulmonary diseases including sarcoidosis and tuberculosis are needed to develop new treatments and biomarkers. Comparing the blood transcriptional response of tuberculosis to other similar pulmonary diseases will advance knowledge of disease pathways and help distinguish diseases with similar clinical presentations. OBJECTIVES: To determine the factors underlying the immunopathogenesis of the granulomatous diseases, sarcoidosis and tuberculosis, by comparing the blood transcriptional responses in these and other pulmonary diseases. METHODS: We compared whole blood genome-wide transcriptional profiles in pulmonary sarcoidosis, pulmonary tuberculosis, to community acquired pneumonia and primary lung cancer and healthy controls, before and after treatment, and in purified leucocyte populations. MEASUREMENTS AND MAIN RESULTS: An Interferon-inducible neutrophil-driven blood transcriptional signature was present in both sarcoidosis and tuberculosis, with a higher abundance and expression in tuberculosis. Heterogeneity of the sarcoidosis signature correlated significantly with disease activity. Transcriptional profiles in pneumonia and lung cancer revealed an over-abundance of inflammatory transcripts. After successful treatment the transcriptional activity in tuberculosis and pneumonia patients was significantly reduced. However the glucocorticoid-responsive sarcoidosis patients showed a significant increase in transcriptional activity. 144-blood transcripts were able to distinguish tuberculosis from other lung diseases and controls. CONCLUSIONS: Tuberculosis and sarcoidosis revealed similar blood transcriptional profiles, dominated by interferon-inducible transcripts, while pneumonia and lung cancer showed distinct signatures, dominated by inflammatory genes. There were also significant differences between tuberculosis and sarcoidosis in the degree of their transcriptional activity, the heterogeneity of their profiles and their transcriptional response to treatment.

Zhang YH, Zhao Y, Li N, Peng YC, Giannoulatou E, Jin RH, Yan HP, Wu H et al. 2013. Interferon-induced transmembrane protein-3 genetic variant rs12252-C is associated with severe influenza in Chinese individuals. Nat Commun, 4 pp. 1418. | Show Abstract | Read more

The SNP rs12252-C allele alters the function of interferon-induced transmembrane protein-3 increasing the disease severity of influenza virus infection in Caucasians, but the allele is rare. However, rs12252-C is much more common in Han Chinese. Here we report that the CC genotype is found in 69% of Chinese patients with severe pandemic influenza A H1N1/09 virus infection compared with 25% in those with mild infection. Specifically, the CC genotype was estimated to confer a sixfold greater risk for severe infection than the CT and TT genotypes. More importantly, because the risk genotype occurs with such a high frequency, its effect translates to a large population-attributable risk of 54.3% for severe infection in the Chinese population studied compared with 5.4% in Northern Europeans. Interferon-induced transmembrane protein-3 genetic variants could, therefore, have a strong effect of the epidemiology of influenza in China and in people of Chinese descent.

Martinez FO, Helming L, Milde R, Varin A, Melgert BN, Draijer C, Thomas B, Fabbri M et al. 2013. Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences. Blood, 121 (9), pp. e57-e69. | Show Abstract | Read more

The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4–activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Although mouse models are widely used for macrophage research, translation to the human can be problematic and the human macrophage system remains poorly described. In the present study, we analyzed and compared the transcriptome and proteome of human and murine macrophages under resting conditions (M0) and after IL-4 activation (M2). We provide a resource for tools enabling macrophage detection in human tissues by identifying a set of 87 macrophage-related genes. Furthermore, we extend current understanding of M2 activation in different species and identify Transglutaminase 2 as a conserved M2 marker that is highly expressed by human macrophages and monocytes in the prototypic Th2 pathology asthma.

Zhao Y, Zhang YH, Denney L, Young D, Powell TJ, Peng YC, Li N, Yan HP et al. 2012. High levels of virus-specific CD4+ T cells predict severe pandemic influenza A virus infection. Am J Respir Crit Care Med, 186 (12), pp. 1292-1297. | Show Abstract | Read more

RATIONALE: T-cell responses have been implicated in control and exacerbation of lung injury during influenza A virus (IAV) infection. OBJECTIVES: To examine the breadth and magnitude of influenza-specific CD4(+) and CD8(+) T-cell responses during acute phase of infection. METHODS: Influenza-specific T-cell response to the entire pandemic H1N1/09 IAV proteome and T cell-related cytokine levels were measured in blood from previously healthy individuals with mild (n = 32) and severe (n = 16) IAV infection during the 2009 influenza pandemic. Virus-specific T-cell response in lung and blood was also performed in two acutely infected, severely ill patients using fluorescent-conjugated pdmH1N1/09 Matrix-MHC-I tetrameric complexes. MEASUREMENTS AND MAIN RESULTS: Strong and broad CD4(+) but not CD8(+) T-cell responses were observed in the blood, and were higher in those with severe disease. Antigen-specific CD8(+) T cells in the lungs were on average 45-fold higher compared with blood in severely ill patients. Paradoxically, in patients with severe disease, IL-17, IL-2, IL-4, and IFN-γ levels were significantly decreased. CONCLUSIONS: High levels of circulating virus-specific CD4(+) T cells to two viral internal proteins (nucleoprotein and matrix) in the first phase of infection are associated with subsequent development of severe IAV infection. This finding could be an early and specific marker for ensuing clinical deterioration. Contrasting levels of antigen-specific CD8(+) T cells in lungs and blood have implications on design and analysis of clinical trials for T-cell vaccines because measurements of T cells in the periphery may not reflect events in the lungs.

Wegmann F, Gartlan KH, Harandi AM, Brinckmann SA, Coccia M, Hillson WR, Kok WL, Cole S et al. 2012. Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens. Nat Biotechnol, 30 (9), pp. 883-888. | Show Abstract | Read more

Protection against mucosally transmitted infections probably requires immunity at the site of pathogen entry, yet there are no mucosal adjuvant formulations licensed for human use. Polyethyleneimine (PEI) represents a family of organic polycations used as nucleic acid transfection reagents in vitro and DNA vaccine delivery vehicles in vivo. Here we show that diverse PEI forms have potent mucosal adjuvant activity for viral subunit glycoprotein antigens. A single intranasal administration of influenza hemagglutinin or herpes simplex virus type-2 (HSV-2) glycoprotein D with PEI elicited robust antibody-mediated protection from an otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen, which were taken up by antigen-presenting cells in vitro and in vivo, promoted dendritic cell trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host double-stranded DNA that triggered Irf3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use.

Denney L, Kok WL, Cole SL, Sanderson S, McMichael AJ, Ho LP. 2012. Activation of invariant NKT cells in early phase of experimental autoimmune encephalomyelitis results in differentiation of Ly6Chi inflammatory monocyte to M2 macrophages and improved outcome. J Immunol, 189 (2), pp. 551-557. | Show Abstract | Read more

Neuropathology in multiple sclerosis is closely linked to presence of macrophages in the CNS. Both M1 (inflammatory) and M2 (alternatively activated, noninflammatory) macrophages are found in the inflamed CNS and thought to differentiate from infiltrating monocytes. It is unclear whether the balance of M1 and M2 macrophages can be altered and whether this affects disease outcome. We show in this article that Ly6C(hi) inflammatory monocytes are the early and dominant infiltrating cells in the CNS during experimental autoimmune encephalomyelitis, a model for the acute phase of multiple sclerosis. Activation of invariant NKT (iNKT) cells reduced the frequency of Ly6C(hi) monocytes and increased the proportion of M2 macrophages in the CNS with associated improvement in neurologic impairment. In contrast, iNKT-deficient mice showed higher numbers of Ly6C(hi) monocytes, reduced M2, and much more severe disease. Adoptive transfer of M2-enriched cells to iNKT-deficient mice markedly improved neurologic impairment. In vitro and in vivo experiments showed that iNKT cells promote differentiation of monocytes to M2 macrophages in an IL-4 and CD1d-dependent process. These findings indicate that infiltrating Ly6C(hi) inflammatory monocytes are early players in acute neuroinflammation and that their frequency and differentiation can be influenced by activation of iNKT cells with resultant improvement in disease outcome.

Everitt AR, Clare S, Pertel T, John SP, Wash RS, Smith SE, Chin CR, Feeley EM et al. 2012. IFITM3 restricts the morbidity and mortality associated with influenza Nature,

Kok WL, Denney L, Benam K, Cole S, Clelland C, McMichael AJ, Ho LP. 2012. Pivotal Advance: Invariant NKT cells reduce accumulation of inflammatory monocytes in the lungs and decrease immune-pathology during severe influenza A virus infection. J Leukoc Biol, 91 (3), pp. 357-368. | Show Abstract | Read more

Little is known of how a strong immune response in the lungs is regulated to minimize tissue injury during severe influenza A virus (IAV) infection. Here, using a model of lethal, high-pathogenicity IAV infection, we first show that Ly6C(hi)Ly6G(-) inflammatory monocytes, and not neutrophils, are the main infiltrate in lungs of WT mice. Mice devoid of iNKT cells (Jα18(-/-) mice) have increased levels of inflammatory monocytes, which correlated with increased lung injury and mortality (but not viral load). Activation of iNKT cells correlated with reduction of MCP-1 levels and improved outcome. iNKT cells were able to selectively lyse infected, MCP-1-producing monocytes in vitro, in a CD1d-dependent process. Our study provides a detailed profile and kinetics of innate immune cells in the lungs during severe IAV infection, highlighting inflammatory monocytes as the major infiltrate and identifying a role for iNKT cells in control of these cells and lung immune-pathology.

Benam KH, Kok WL, McMichael AJ, Ho LP. 2011. Alternative spliced CD1d transcripts in human bronchial epithelial cells PLoS ONE, 6 (8), | Show Abstract | Read more

CD1d is a MHC I like molecule which presents glycolipid to natural killer T (NKT) cells, a group of cells with diverse but critical immune regulatory functions in the immune system. These cells are required for optimal defence against bacterial, viral, protozoan, and fungal infections, and control of immune-pathology and autoimmune diseases. CD1d is expressed on antigen presenting cells but also found on some non-haematopoietic cells. However, it has not been observed on bronchial epithelium, a site of active host defence in the lungs. Here, we identify for the first time, CD1D mRNA variants and CD1d protein expression on human bronchial epithelial cells, describe six alternatively spliced transcripts of this gene in these cells; and show that these variants are specific to epithelial cells. These findings provide the basis for investigations into a role for CD1d in lung mucosal immunity. © 2011 Benam et al.

Lockstone HE, Sanderson S, Kulakova N, Baban D, Leonard A, Kok WL, McGowan S, McMichael AJ, Ho LP. 2010. Gene set analysis of lung samples provides insight into pathogenesis of progressive, fibrotic pulmonary sarcoidosis. Am J Respir Crit Care Med, 181 (12), pp. 1367-1375. | Show Abstract | Read more

RATIONALE: Approximately 60 to 70% of patients with pulmonary sarcoidosis have disease that resolves spontaneously; the rest follow a chronic course with varying levels of fibrosis. It is unclear why some patients progress and if treatment affects outcome. OBJECTIVES: To determine differential gene expression profile in lungs of patients with self-limiting sarcoidosis compared to those with progressive-fibrotic disease, and to analyze the biological relevance of these differentially expressed genes. METHODS: We examined microarray expression of 26,626 genes in transbronchial biopsies of granulomatous areas in lungs of patients with active but self-limiting (n = 8) versus those with active, progressive (+/- fibrotic) pulmonary disease (n = 7). MEASUREMENTS AND MAIN RESULTS: Three hundred thirty-four genes were differentially expressed between the two groups (P < 0.01, Bayesian moderated t test). Gene Set Enrichment Analysis showed over-representation of gene-sets (defined by Gene Ontology) related to host immune activation, proliferation, and defense, among genes up-regulated in the progressive-fibrotic group (FDR q < 0.0001 for the top 43 gene sets), and a marked enrichment of, and similarity in gene expression profiles between, progressive-fibrotic sarcoidosis and hypersensitivity pneumonitis (HP), (q < 0.001), but not idiopathic pulmonary fibrosis (IPF). CONCLUSIONS: The findings suggest that patients with progressive/fibrotic pulmonary sarcoidosis have intense immune activity related to host defense in their lungs, with processes more similar to HP than IPF. The study also demonstrates that transbronchial lung biopsy samples can provide good-quality RNA for gene expression profiling, supporting its potential use as a prognostic classifier for pulmonary sarcoidosis.

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