Identification of an immunogenic 18 kDa protein of Helicobacter pylori using alkaline phosphatase gene fusions : a thesis presented in partial fulfillment of the requirement for the Doctor of Philosophy in Molecular Microbiology, Massey University, Palmerston North, NZ

Secreted or surface-associated proteins play an important role in the immunopathogenesis of Helicobacter pylori infection. The aim of this study was to identify, using a genetic approach, H. pylori exported proteins and assess their role in the host immune response to infection. As part of this work, an H. pylori expression library was constructed and screened with a monoclonal antibody raised to a component of outer membrane vesicles from H. pylori, identified and characterised in a separate study. The screening strategy identified a locus of the genome containing two genes encoding exported proteins. Subsequent expression studies identified the gene product detected by the antibody as Lpp20, which encodes a well characterised lipoprotein from H. pylori. In addition, the use of alkaline phosphatase (AP) gene fusion methodology enabled the identification of a large number of other H. pylori exported proteins. Immunoscreening of a selection of enzymatically active H. pylori AP fusion proteins was carried out by Western blot analysis with patient sera and lymphocyte proliferation assays using peripheral blood mononuclear cells from H. pylori infected individuals. These assays identified a novel H. pylori exported antigen which was recognised by antibody derived from H. pylori infected individuals. Southern blot analysis revealed that the gene encoding the protein was absent in other Helicobacter species tested and sequence analysis of the gene demonstrated that it is highly conserved among H. pylori isolates. In order to obtain pure recombinant protein, the gene encoding the protein was cloned and expressed as a Beta-galactosidase (β-gal) fusion in Escherichia coli and the protein purified by affinity chromatography. The size of the recombinant protein released (18 kDa) was consistent with the calculated molecular mass of the polypeptide deduced from the DNA sequence. In Western blot assays, the purified protein was recognised by 71% of sera taken from patients infected with H. pylori, but by only 16% of sera taken from patients with unrelated or with no gastrointestinal disease. These results indicated that the 18 kDa protein from H. pylori was immunogenic and expressed in vivo. In other experiments, it was found that oral administration of this antigen did not protect mice against H. pylori colonisation following challenge with H. pylori

Interplay of Polarity Proteins and GTPases in T-Lymphocyte Function

Polarity refers to the asymmetric distribution of different cellular components within a cell and is central to many cell functions. In T-cells, polarity regulates the activation, migration, and effector function of cytotoxic T-cells (CTLs) during an immune response. The regulation of asymmetric cell division by polarity proteins may also dictate CTL effector and memory differentiation following antigen presentation. Small GTPases, along with their associated polarity and adaptor proteins, are critical for mediating the polarity changes necessary for T-cell activation and function, and in turn, are regulated by guanine exchange factors (GEFS) and GTPase activating proteins (GAPS). For example, a novel GEF, dedicator of cytokinesis 8 (DOCK8) was recently identified as a regulator of immune cell function and mutations in DOCK8 have been detected in patients with severe combined immunodeficiency. Both B and T-cells from DOCK8 mutant mice form defective immunological synapses and have abnormal functions, in addition to impaired immune memory development. This paper will discuss the interplay between polarity proteins and GTPases, and their role in T-cell function

Interplay of Polarity Proteins and GTPases in T-Lymphocyte Function

Polarity refers to the asymmetric distribution of different cellular components within a cell and is central to many cell functions. In T-cells, polarity regulates the activation, migration, and effector function of cytotoxic T-cells (CTLs) during an immune response. The regulation of asymmetric cell division by polarity proteins may also dictate CTL effector and memory differentiation following antigen presentation. Small GTPases, along with their associated polarity and adaptor proteins, are critical for mediating the polarity changes necessary for T-cell activation and function, and in turn, are regulated by guanine exchange factors (GEFS) and GTPase activating proteins (GAPS). For example, a novel GEF, dedicator of cytokinesis 8 (DOCK8) was recently identified as a regulator of immune cell function and mutations in DOCK8 have been detected in patients with severe combined immunodeficiency. Both B and T-cells from DOCK8 mutant mice form defective immunological synapses and have abnormal functions, in addition to impaired immune memory development. This paper will discuss the interplay between polarity proteins and GTPases, and their role in T-cell function

Interplay of Polarity Proteins and GTPases in T-Lymphocyte Function

Polarity refers to the asymmetric distribution of different cellular components within a cell and is central to many cell functions. In T-cells, polarity regulates the activation, migration, and effector function of cytotoxic T-cells (CTLs) during an immune response. The regulation of asymmetric cell division by polarity proteins may also dictate CTL effector and memory differentiation following antigen presentation. Small GTPases, along with their associated polarity and adaptor proteins, are critical for mediating the polarity changes necessary for T-cell activation and function, and in turn, are regulated by guanine exchange factors (GEFS) and GTPase activating proteins (GAPS). For example, a novel GEF, dedicator of cytokinesis 8 (DOCK8) was recently identified as a regulator of immune cell function and mutations in DOCK8 have been detected in patients with severe combined immunodeficiency. Both B and T-cells from DOCK8 mutant mice form defective immunological synapses and have abnormal functions, in addition to impaired immune memory development. This paper will discuss the interplay between polarity proteins and GTPases, and their role in T-cell function

The Reorientation of T-Cell Polarity and Inhibition of Immunological Synapse Formation by CD46 Involves Its Recruitment to Lipid Rafts

Many infectious agents utilize CD46 for infection of human cells, and therapeutic applications of CD46-binding viruses are now being explored. Besides mediating internalization to enable infection, binding to CD46 can directly alter immune function. In particular, ligation of CD46 by antibodies or by measles virus can prevent activation of T cells by altering T-cell polarity and consequently preventing the formation of an immunological synapse. Here, we define a mechanism by which CD46 reorients T-cell polarity to prevent T-cell receptor signaling in response to antigen presentation. We show that CD46 associates with lipid rafts upon ligation, and that this reduces recruitment of both lipid rafts and the microtubule organizing centre to the site of receptor cross-linking. These data combined indicate that polarization of T cells towards the site of CD46 ligation prevents formation of an immunological synapse, and this is associated with the ability of CD46 to recruit lipid rafts away from the site of TCR ligation

NKG7 enhances CD8+ T cell synapse efficiency to limit inflammation

Cytotoxic lymphocytes are essential for anti-tumor immunity, and for effective responses to cancer immunotherapy. Natural killer cell granule protein 7 (NKG7) is expressed at high levels in cytotoxic lymphocytes infiltrating tumors from patients treated with immunotherapy, but until recently, the role of this protein in cytotoxic lymphocyte function was largely unknown. Unexpectedly, we found that highly CD8+ T cell-immunogenic murine colon carcinoma (MC38-OVA) tumors grew at an equal rate in Nkg7+/+ and Nkg7-/- littermate mice, suggesting NKG7 may not be necessary for effective CD8+ T cell anti-tumor activity. Mechanistically, we found that deletion of NKG7 reduces the ability of CD8+ T cells to degranulate and kill target cells in vitro. However, as a result of inefficient cytotoxic activity, NKG7 deficient T cells form a prolonged immune synapse with tumor cells, resulting in increased secretion of inflammatory cytokines, including tumor necrosis factor alpha (TNF). By deleting the TNF receptor, TNFR1, from MC38-OVA tumors, we demonstrate that this hyper-secretion of TNF compensates for reduced synapse-mediated cytotoxic activity against MC38-OVA tumors in vivo, via increased TNF-mediated tumor cell death. Taken together, our results demonstrate that NKG7 enhances CD8+ T cell immune synapse efficiency, which may serve as a mechanism to accelerate direct cytotoxicity and limit potentially harmful inflammatory responses

Protection from EAE in DOCK8 mutant mice occurs despite increased Th17 cell frequencies in the periphery.

peer reviewedMutation of Dedicator of cytokinesis 8 (DOCK8) has previously been reported to provide resistance to the Th17 cell dependent EAE in mice. Contrary to expectation, we observed an elevation of Th17 cells in two different DOCK8 mutant mouse strains in the steady state. This was specific for Th17 cells with no change in Th1 or Th2 cell populations. In vitro Th cell differentiation assays revealed that the elevated Th17 cell population was not due to a T cell intrinsic differentiation bias. Challenging these mutant mice in the EAE model, we confirmed a resistance to this autoimmune disease with Th17 cells remaining elevated systemically while cellular infiltration in the CNS was reduced. Infiltrating T cells lost the bias toward Th17 cells indicating a relative reduction of Th17 cells in the CNS and a Th17 cell specific migration disadvantage. Adoptive transfers of Th1 and Th17 cells in EAE-affected mice further supported the Th17 cell-specific migration defect, however, DOCK8-deficient Th17 cells expressed normal Th17 cell-specific CCR6 levels and migrated toward chemokine gradients in transwell assays. This study shows that resistance to EAE in DOCK8 mutant mice is achieved despite a systemic Th17 bias

DOCK8 deficiency impairs CD8 T cell survival and function in humans and mice

In humans, DOCK8 immunodeficiency syndrome is characterized by severe cutaneous viral infections. Thus, CD8 T cell function may be compromised in the absence of DOCK8. In this study, by analyzing mutant mice and humans, we demonstrate a critical, intrinsic role for DOCK8 in peripheral CD8 T cell survival and function. DOCK8 mutation selectively diminished the abundance of circulating naive CD8 T cells in both species, and in DOCK8-deficient humans, most CD8 T cells displayed an exhausted CD45RA+CCR7? phenotype. Analyses in mice revealed the CD8 T cell abnormalities to be cell autonomous and primarily postthymic. DOCK8 mutant naive CD8 T cells had a shorter lifespan and, upon encounter with antigen on dendritic cells, exhibited poor LFA-1 synaptic polarization and a delay in the first cell division. Although DOCK8 mutant T cells underwent near-normal primary clonal expansion after primary infection with recombinant influenza virus in vivo, they showed greatly reduced memory cell persistence and recall. These findings highlight a key role for DOCK8 in the survival and function of human and mouse CD8 T cells

DOCK8 deficiency impairs CD8 T cell survival and function in humans and mice

As shown by analysis of mice and humans bearing DOCK8-inactivating mutations, DOCK8 plays a cell-autonomous role in survival of naive CD8 T cells, LFA-1 polarization toward the immune synapse, and CD8 T cell memory and recall responses following viral infection

Non-invasive diagnostic tests for Helicobacter pylori infection

BACKGROUND: Helicobacter pylori (H pylori) infection has been implicated in a number of malignancies and non-malignant conditions including peptic ulcers, non-ulcer dyspepsia, recurrent peptic ulcer bleeding, unexplained iron deficiency anaemia, idiopathic thrombocytopaenia purpura, and colorectal adenomas. The confirmatory diagnosis of H pylori is by endoscopic biopsy, followed by histopathological examination using haemotoxylin and eosin (H & E) stain or special stains such as Giemsa stain and Warthin-Starry stain. Special stains are more accurate than H & E stain. There is significant uncertainty about the diagnostic accuracy of non-invasive tests for diagnosis of H pylori. OBJECTIVES: To compare the diagnostic accuracy of urea breath test, serology, and stool antigen test, used alone or in combination, for diagnosis of H pylori infection in symptomatic and asymptomatic people, so that eradication therapy for H pylori can be started. SEARCH METHODS: We searched MEDLINE, Embase, the Science Citation Index and the National Institute for Health Research Health Technology Assessment Database on 4 March 2016. We screened references in the included studies to identify additional studies. We also conducted citation searches of relevant studies, most recently on 4 December 2016. We did not restrict studies by language or publication status, or whether data were collected prospectively or retrospectively. SELECTION CRITERIA: We included diagnostic accuracy studies that evaluated at least one of the index tests (urea breath test using isotopes such as13C or14C, serology and stool antigen test) against the reference standard (histopathological examination using H & E stain, special stains or immunohistochemical stain) in people suspected of having H pylori infection. DATA COLLECTION AND ANALYSIS: Two review authors independently screened the references to identify relevant studies and independently extracted data. We assessed the methodological quality of studies using the QUADAS-2 tool. We performed meta-analysis by using the hierarchical summary receiver operating characteristic (HSROC) model to estimate and compare SROC curves. Where appropriate, we used bivariate or univariate logistic regression models to estimate summary sensitivities and specificities. MAIN RESULTS: We included 101 studies involving 11,003 participants, of which 5839 participants (53.1%) had H pylori infection. The prevalence of H pylori infection in the studies ranged from 15.2% to 94.7%, with a median prevalence of 53.7% (interquartile range 42.0% to 66.5%). Most of the studies (57%) included participants with dyspepsia and 53 studies excluded participants who recently had proton pump inhibitors or antibiotics.There was at least an unclear risk of bias or unclear applicability concern for each study.Of the 101 studies, 15 compared the accuracy of two index tests and two studies compared the accuracy of three index tests. Thirty-four studies (4242 participants) evaluated serology; 29 studies (2988 participants) evaluated stool antigen test; 34 studies (3139 participants) evaluated urea breath test-13C; 21 studies (1810 participants) evaluated urea breath test-14C; and two studies (127 participants) evaluated urea breath test but did not report the isotope used. The thresholds used to define test positivity and the staining techniques used for histopathological examination (reference standard) varied between studies. Due to sparse data for each threshold reported, it was not possible to identify the best threshold for each test.Using data from 99 studies in an indirect test comparison, there was statistical evidence of a difference in diagnostic accuracy between urea breath test-13C, urea breath test-14C, serology and stool antigen test (P = 0.024). The diagnostic odds ratios for urea breath test-13C, urea breath test-14C, serology, and stool antigen test were 153 (95% confidence interval (CI) 73.7 to 316), 105 (95% CI 74.0 to 150), 47.4 (95% CI 25.5 to 88.1) and 45.1 (95% CI 24.2 to 84.1). The sensitivity (95% CI) estimated at a fixed specificity of 0.90 (median from studies across the four tests), was 0.94 (95% CI 0.89 to 0.97) for urea breath test-13C, 0.92 (95% CI 0.89 to 0.94) for urea breath test-14C, 0.84 (95% CI 0.74 to 0.91) for serology, and 0.83 (95% CI 0.73 to 0.90) for stool antigen test. This implies that on average, given a specificity of 0.90 and prevalence of 53.7% (median specificity and prevalence in the studies), out of 1000 people tested for H pylori infection, there will be 46 false positives (people without H pylori infection who will be diagnosed as having H pylori infection). In this hypothetical cohort, urea breath test-13C, urea breath test-14C, serology, and stool antigen test will give 30 (95% CI 15 to 58), 42 (95% CI 30 to 58), 86 (95% CI 50 to 140), and 89 (95% CI 52 to 146) false negatives respectively (people with H pylori infection for whom the diagnosis of H pylori will be missed).Direct comparisons were based on few head-to-head studies. The ratios of diagnostic odds ratios (DORs) were 0.68 (95% CI 0.12 to 3.70; P = 0.56) for urea breath test-13C versus serology (seven studies), and 0.88 (95% CI 0.14 to 5.56; P = 0.84) for urea breath test-13C versus stool antigen test (seven studies). The 95% CIs of these estimates overlap with those of the ratios of DORs from the indirect comparison. Data were limited or unavailable for meta-analysis of other direct comparisons. AUTHORS' CONCLUSIONS: In people without a history of gastrectomy and those who have not recently had antibiotics or proton ,pump inhibitors, urea breath tests had high diagnostic accuracy while serology and stool antigen tests were less accurate for diagnosis of Helicobacter pylori infection.This is based on an indirect test comparison (with potential for bias due to confounding), as evidence from direct comparisons was limited or unavailable. The thresholds used for these tests were highly variable and we were unable to identify specific thresholds that might be useful in clinical practice.We need further comparative studies of high methodological quality to obtain more reliable evidence of relative accuracy between the tests. Such studies should be conducted prospectively in a representative spectrum of participants and clearly reported to ensure low risk of bias. Most importantly, studies should prespecify and clearly report thresholds used, and should avoid inappropriate exclusions