Wafa Khamri

Role of expression of the tumour-associated macrophage receptor, MERTK, in hepatocellular carcinoma: Spring Meeting for Clinician Scientists in Training 2017

BACKGROUND: Venous thromboembolism is a common, potentially avoidable cause of death and morbidity in patients in hospital, including those with stroke. In surgical patients, intermittent pneumatic compression (IPC) reduces the risk of deep vein thrombosis (DVT), but no reliable evidence exists about its effectiveness in patients who have had a stroke. We assessed the effectiveness of IPC to reduce the risk of DVT in patients who have had a stroke. METHODS: The CLOTS 3 trial is a multicentre parallel group randomised trial assessing IPC in immobile patients (ie, who cannot walk to the toilet without the help of another person) with acute stroke. We enrolled patients from day 0 to day 3 of admission and allocated them via a central randomisation system (ratio 1:1) to receive either IPC or no IPC. A technician who was masked to treatment allocation did a compression duplex ultrasound (CDU) of both legs at 7-10 days and, wherever practical, at 25-30 days after enrolment. Caregivers and patients were not masked to treatment assignment. Patients were followed up for 6 months to determine survival and later symptomatic venous thromboembolism. The primary outcome was a DVT in the proximal veins detected on a screening CDU or any symptomatic DVT in the proximal veins, confirmed on imaging, within 30 days of randomisation. Patients were analysed according to their treatment allocation. TRIAL REGISTRATION: ISRCTN93529999. FINDINGS: Between Dec 8, 2008, and Sept 6, 2012, 2876 patients were enrolled in 94 centres in the UK. The included patients were broadly representative of immobile stroke patients admitted to hospital and had a median age of 76 years (IQR 67-84). The primary outcome occurred in 122 (8·5%) of 1438 patients allocated IPC and 174 (12·1%) of 1438 patients allocated no IPC; an absolute reduction in risk of 3·6% (95% CI 1·4-5·8). Excluding the 323 patients who died before any primary outcome and 41 without any screening CDU, the adjusted OR for the comparison of 122 of 1267 patients vs 174 of 1245 patients was 0·65 (95% CI 0·51-0·84; p=0·001). Deaths in the treatment period occurred in 156 (11%) patients allocated IPC and 189 (13%) patients allocated no IPC died within the 30 days of treatment period (p=0·057); skin breaks on the legs were reported in 44 (3%) patients allocated IPC and in 20 (1%) patients allocated no IPC (p=0·002); falls with injury were reported in 33 (2%) patients in the IPC group and in 24 (2%) patients in the no-IPC group (p=0·221). INTERPRETATION: IPC is an effective method of reducing the risk of DVT and possibly improving survival in a wide variety of patients who are immobile after stroke. FUNDING: National Institute of Health Research (NIHR) Health Technology Assessment (HTA) programme, UK; Chief Scientist Office of Scottish Government; Covidien (MA, USA).

CD14, CD16 and HLA-DR reliably identifies human monocytes and their subsets in the context of pathologically reduced HLA-DR expression by CD14(hi) /CD16(neg) monocytes: Expansion of CD14(hi) /CD16(pos) and contraction of CD14(lo) /CD16(pos) monocytes in acute liver failure

Changes in monocytes and their subsets (CD14hi/CD16neg, CD14hi/CD16pos and CD14lo/CD16pos) have been described in several diseases. The combination of CD14, CD16 and HLA‐DR has been suggested to discriminate monocytes from the CD16pos/HLA‐DRneg NK‐cells and neutrophils but no data exist whether this strategy can be used in situations when monocyte HLA‐DR expression is pathologically reduced. Monocytes and their subsets were concurrently identified through negative (exclusion of CD66bpos neutrophils, CD56pos NKcells, CD19pos B‐cells, and CD3pos T‐cells) and positive gating (inclusion of monocytes by expression of CD14, CD16, and HLA‐DR) strategies on 30 occasions [9 healthy controls (HC) and 21 patients with conditions associated with low monocyte HLA‐DR expression]. Bland‐Altman and Passing and Bablok regression statistics did not demonstrate any significant measurement bias between the two strategies of monocyte identification. Monocyte subset phenotype was then compared in 18 HC and 41 patients with acute liver failure (ALF). Compared with HC, in ALF, the percentage of CD14hi/CD16pos monocytes was higher (7% vs 4%) whilst the percentage of CD14lo/CD16pos was lower (1.9% vs. 7%) (P ≤ 0.001); HLA‐DR and CD86 MFIs on all monocyte subsets were lower, whilst CCR5, CD64, and CD11b MFIs were higher (P < 0.05). The relative expression by monocyte subsets of HLA‐DR, CCR2, CCR5, CX3CR1, and CD11a was similar in ALF patients and HCs. Repeat analysis of an identical antibody‐fluorochrome “backbone” targeting HLA‐DR, CD14, and CD16 was assessed in 189 samples across 5 different experiments. There was excellent agreement in the results obtained using the positive gating strategy (interclass correlation coefficients > 0.8). Monocytes and their subsets can be reliably identified using an antibody‐fluorochrome “backbone” of HLA‐DR, CD14, and CD16. CD16pos monocytes continue to constitutively express HLA‐DR even in conditions where HLA‐DR is pathologically reduced on CD14hi/CD16neg monocytes. Understanding the changes in monocyte pheontype in ALF and similar clinico‐pathological diseases may allow the development of novel biomarkers or therapeutic strategies.

Chronic Exposure to Helicobacter pylori Impairs Dendritic Cell Function and Inhibits Th1 Development

ABSTRACT Helicobacter pylori causes chronic gastric infection that affects the majority of the worlds population. Despite generating an inflammatory response, the immune system usually fails to clear the infection. Since dendritic cells (DCs) play a pivotal role in shaping the immune response, we investigated the effects of H. pylori on DC function. We have demonstrated that H. pylori increased the expression of activation markers on DCs while upregulating the inhibitory B7 family molecule, PD-L1. Functionally, H. pylori-treated DCs resulted in the production of interleukin-10 (IL-10) and IL-23 but not of alpha interferon (IFN-α). While very little or no IL-12 was produced to H. pylori alone, simultaneous ligation of CD40 on DCs induced IL-12 release. We also demonstrated that DCs treated with H. pylori-induced IFN-γ production by allogeneic naive T cells. However, stimulation of DCs with H. pylori for an extended period of time impaired their ability to produce cytokines after CD40 ligation and limited their ability to promote IFN-γ release, suggesting that the DCs had become exhausted by the prolonged stimulation. The effect of chronic infection with H. pylori on DC function was further investigated by focusing on DC development. Demonstrating that monocytes differentiated into DCs in the presence of H. pylori exhibited an exhausted phenotype with an impaired ability to produce IL-12 and a downregulation of CD1a. Our results raise the possibility that in chronic H. pylori infection DCs become exhausted after prolonged antigen exposure leading to suboptimal Th1 development. This effect may contribute to persistence of H. pylori infection.

CD161 expression characterizes a subpopulation of human regulatory T cells that produces IL‐17 in a STAT3‐dependent manner

Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell‐based clinical therapy. However, human Treg cells are “plastic”, and are able to produce IL‐17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL‐17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL‐17 in vitro when activated in the presence of IL‐1β, but not IL‐6. “IL‐17 potential” is restricted to population III (CD4+CD25hiCD127loCD45RA−) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL‐17 induction. Importantly, we find that IL‐17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL‐17. Finally, we show that CD161+ population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL‐17‐producing Treg‐cell population at these sites. As IL‐17 production from this Treg‐cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.

Helicobacter pylori Stimulates Dendritic Cells To Induce Interleukin-17 Expression from CD4+ T Lymphocytes

ABSTRACT Helicobacter pylori is a human gastroduodenal pathogen that leads to active chronic inflammation characterized by T-cell responses biased toward a Th1 phenotype. It has been accepted that H. pylori infection induces a Th17 response. At mucosal sites, dendritic cells (DCs) have the capacity to induce effector T cells. Here, we evaluate the role of DCs in the H. pylori-induced interleukin-17 (IL-17) response. Immunohistochemistry and immunofluorescence were performed on human gastric mucosal biopsy samples and showed that myeloid DCs in H. pylori-infected patients colocalized with IL-23- and that IL-17-producing lymphocytes were present in H. pylori-infected antral biopsy samples. In parallel, human monocyte-derived DCs stimulated in vitro with live H. pylori cells produced significant levels of IL-23 in the absence of IL-12 release. The subsequent incubation of H. pylori-infected DCs with autologous CD4+ T cells led to gamma interferon (IFN-γ) and IL-17 expression. The inhibition of IL-1 and, to a lesser extent, IL-23 inhibited IL-17 production by T cells. Finally, isogenic H. pylori mutant strains not expressing major virulence factors were less effective in inducing IL-1 and IL-23 release by DCs and IL-17 release by T cells than parental strains. Altogether, we can conclude that DCs are potent inducers of IL-23/IL-17 expression following H. pylori stimulation. IL-1/IL-23 as well as H. pylori virulence factors seem to play an important role in mediating this response.

Expression of complement components, receptors and regulators by human dendritic cells.

Integration of innate and adaptive arms of the immune response at a cellular and molecular level appears to be fundamental to the development of powerful effector functions in host defence and aberrant immune responses. Here we provide evidence that the functions of human complement activation and antigen presentation converge on dendritic cells (DCs). We show that several subsets of human DCs [i.e., monocyte derived (CD1a+CD14−), dermal (CD1a+DC-SIGN+), Langerhans (CD1a+Langerin+), myeloid (CD1c+CD19−), plamacytoid (CD45RA+CD123+)] express many of the components of the classical and alternative and terminal pathways of complement. Moreover human DCs have receptors known to detect the biologically active peptides C3a and C5a (C3aR, C5aR) and the covalently bound fragments C3b and metabolites iC3b and C3d which serve in immune adhesion (i.e., CR3, CR4, CRIg). We also show that the human DC surface is characterised by membrane bound regulators of complement activation, which are also known to participate in intracellular signalling (i.e., CD46, CD55, CD59). This work provides an extensive description of complement components relevant to the integrated actions of complement and DC, illuminated by animal studies. It acts as a resource that allows further understanding and exploitation of role of complement in human health and immune mediated diseases.

Expression of Surfactant Protein D in the Human Gastric Mucosa and during Helicobacter pylori Infection

ABSTRACT Helicobacter pylori establishes persistent infection of gastric mucosa with diverse clinical outcomes. The innate immune molecule surfactant protein D (SP-D) binds selectively to microorganisms, inducing aggregation and phagocytosis. In this study, we demonstrated the expression of SP-D in gastric mucosa by reverse transcription-PCR and immuohistochemical analysis. SP-D is present at the luminal surface and within the gastric pits, with maximal expression at the surface. Levels of expression are significantly increased in H. pylori-associated gastritis compared to those in the normal mucosa. Immunofluorescence microscopy was used to demonstrate binding and agglutination of H. pylori by SP-D in a lectin-specific manner. These activities resulted in a 50% reduction in the motility of H. pylori, as judged on the basis of curvilinear velocity measured by using a Hobson BacTracker. Lipopolysaccharides extracted from three H. pylori strains were shown to bind SP-D in a concentration-dependent manner, and there was marked variation in the avidity of binding among the strains. SP-D may therefore play a significant role in the innate immune response to H. pylori infection.

Variations in Helicobacter pylori Lipopolysaccharide To Evade the Innate Immune Component Surfactant Protein D

ABSTRACT Helicobacter pylori is a common and persistent human pathogen of the gastric mucosa. Surfactant protein D (SP-D), a component of innate immunity, is expressed in the human gastric mucosa and is capable of aggregating H. pylori. Wide variation in the SP-D binding affinity to H. pylori has been observed in clinical isolates and laboratory-adapted strains. The aim of this study was to reveal potential mechanisms responsible for evading SP-D binding and establishing persistent infection. An escape variant, J178V, was generated in vitro, and the lipopolysaccharide (LPS) structure of the variant was compared to that of the parental strain, J178. The genetic basis for structural variation was explored by sequencing LPS biosynthesis genes. SP-D binding to clinical isolates was demonstrated by fluorescence-activated cell sorter analyses. Here, we show that H. pylori evades SP-D binding through phase variation in lipopolysaccharide. This phenomenon is linked to changes in the fucosylation of the O chain, which was concomitant with slipped-strand mispairing in a poly(C) tract of the fucosyltransferase A (fucT1) gene. SP-D binding organisms are predominant in mucus in vivo (P = 0.02), suggesting that SP-D facilitates physical elimination. Phase variation to evade SP-D contributes to the persistence of this common gastric pathogen.

New chromogenic agar medium for the identification of Candida spp.

ABSTRACT A new chromogenic agar medium (Candida diagnostic agar [CDA]) for differentiation of Candida spp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-{2-[4-(2-acetamido-2-deoxy-β-d-glucopyranosyloxy)-3-methoxyphenyl]-vinyl}-1-(propan-3-yl-oate)-quinolium bromide (0.32 g liter−1). The glucosaminidase substrate in CDA was hydrolyzed by Candida albicans and Candida dubliniensis, yielding white colonies with deep-red spots on a yellow transparent background after 24 to 48 h of incubation at 37°C. Colonies of Candida tropicalis and Candida kefyr were uniformly pink, and colonies of other Candida spp., including Candida glabrata and Candida parapsilosis, were white. CDA was evaluated by using 115 test strains of Candida spp. and other clinically important yeasts and was compared with two commercially available chromogenic agars (Candida ID agar [bioMerieux] and CHROMagar Candida [CHROMagar Company Ltd.]). On all three agars, colonies of C. albicans were not distinguished from colonies of C. dubliniensis. However, for the group containing C. albicans plus C. dubliniensis, both the sensitivity and the specificity of detection when CDA was used were 100%, compared with values of 97.6 and 100%, respectively, with CHROMagar Candida and 100 and 96.8%, respectively, with Candida ID agar. In addition, for the group containing C. tropicalis plus C. kefyr, the sensitivity and specificity of detection when CDA was used were also 100%, compared with 72.7 and 98.1%, respectively, with CHROMagar Candida. Candida ID agar did not differentiate C. tropicalis and C. kefyr strains but did differentiate members of a broader group (C. tropicalis, C. kefyr, Candida lusitaniae plus Candida guilliermondii); the sensitivity and specificity of detection for members of this group were 94.7 and 93.8%, respectively. In addition to the increased sensitivity and/or specificity of Candida detection when CDA was used, differentiation of colony types on CDA (red spotted, pink, or no color) was unambiguous and did not require precise assessment of colony color.

Secretory leukocyte protease inhibitor: a pivotal mediator of anti-inflammatory responses in acetaminophen-induced acute liver failure.

Acetaminophen‐induced acute liver failure (AALF) is characterized both by activation of innate immune responses and susceptibility to sepsis. Circulating monocytes and hepatic macrophages are central mediators of inflammatory responses and tissue repair processes during human AALF. Secretory leukocyte protease inhibitor (SLPI) modulates monocyte/macrophage function through inhibition of nuclear factor kappa B (NF‐κB) signaling. The aims of this study were to establish the role of SLPI in AALF. Circulating levels of SLPI, monocyte cluster of differentiation 163 (CD163), human leukocyte antigen‐DR (HLA‐DR), and lipopolysaccharide (LPS)‐stimulated levels of NF‐κBp65, tumor necrosis factor alpha (TNF‐α) and interleukin (IL)‐6 were determined in patients with AALF, chronic liver disease, and healthy controls. Immunohistochemistry and multispectral imaging of AALF explant tissue determined the cellular sources of SLPI and hepatic macrophage phenotype. The phenotype and function of monocytes and macrophages was determined following culture with recombinant human (rh)‐SLPI, liver homogenates, and plasma derived from AALF patients in the presence and absence of antihuman (α)SLPI. Hepatic and circulatory concentrations of SLPI were elevated in AALF and immunohistochemistry revealed SLPI expression in biliary epithelial cells and within hepatic macrophages (h‐mψ) in areas of necrosis. H‐mψ and circulating monocytes in AALF exhibited an anti‐inflammatory phenotype and functional characteristics; typified by reductions in NF‐κBp65, TNF‐α, and IL‐6 and preserved IL‐10 secretion following LPS challenge. Culture of healthy monocytes with AALF liver homogenates, plasma, or rhSLPI induced monocytes with strikingly similar anti‐inflammatory characteristics which were reversed by inhibiting the activity of SLPI. Conclusion: SLPI is a pivotal mediator of anti‐inflammatory responses in AALF through modulation of monocyte/macrophage function, which may account for the susceptibility to sepsis in AALF. (Hepatology 2014;59:1564‐1576)