Holly Stephenson

Selective ERK Activation Differentiates Mouse and Human Tolerogenic Dendritic Cells, Expands Antigen-Specific Regulatory T Cells, and Suppresses Experimental Inflammatory Arthritis

Objective Most therapeutic treatments for autoimmune arthritis rely on immunosuppressive drugs, which have side effects. Although a previous study by our group showed that specific ERK activation suppressed immune responses, its application in a therapeutic setting has never been tested. The aim of the present study was to define the ERK-dependent immunosuppressive mechanisms and to apply selective ERK activation for the treatment of experimental inflammatory arthritis. Methods A constitutively active ERK activator was coexpressed with a model antigen using lentivectors. Immunosuppressive mechanisms were characterized at the level of dendritic cell (DC) function, differentiation of antigen-specific Treg cells, and inhibition of inflammatory T cells. Administration of the ERK activator with antigen as a strategy to suppress inflammatory arthritis was tested in an experimental mouse model. Results Selective ERK activation induced mouse and human DCs to secrete bioactive transforming growth factor β, a process required for suppression of T cell responses and differentiation of antigen-specific Treg cells. Treg cells strongly proliferated after antigen reencounter in inflammatory conditions, and these cells exhibited antigen-dependent suppressive activities. Inflammatory arthritis was effectively inhibited through antigen-specific mechanisms. Importantly, this strategy did not rely on identification of the initiating arthritogenic antigen. Equivalent mechanisms were demonstrated in human monocyte–derived DCs, setting the scene for a possible rapid translation of this approach to patients with rheumatoid arthritis. Conclusion This strategy of selective ERK activation resulted in an effective therapeutic protocol, with substantial advantages over DC or T cell vaccination.

Delineation of the Innate and Adaptive T-Cell Immune Outcome in the Human Host in Response to Campylobacter jejuni Infection

Background Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Despite the significant health burden this infection presents, molecular understanding of C. jejuni-mediated disease pathogenesis remains poorly defined. Here, we report the characterisation of the early, innate immune response to C. jejuni using an ex-vivo human gut model of infection. Secondly, impact of bacterial-driven dendritic cell activation on T-cell mediated immunity was also sought. Methodology Healthy, control paediatric terminal ileum or colonic biopsy tissue was infected with C. jejuni for 8–12 hours. Bacterial colonisation was followed by confocal microscopy and mucosal innate immune responses measured by ELISA. Marked induction of IFNγ with modest increase in IL-22 and IL-17A was noted. Increased mucosal IL-12, IL-23, IL-1β and IL-6 were indicative of a cytokine milieu that may modulate subsequent T-cell mediated immunity. C. jejuni-driven human monocyte-derived dendritic cell activation was followed by analyses of T cell immune responses utilising flow cytometry and ELISA. Significant increase in Th-17, Th-1 and Th-17/Th-1 double-positive cells and corresponding cytokines was observed. The ability of IFNγ, IL-22 and IL-17 cytokines to exert host defence via modulation of C. jejuni adhesion and invasion to intestinal epithelia was measured by standard gentamicin protection assay. Conclusions Both innate and adaptive T cell-immunity to C. jejuni infection led to the release of IFNγ, IL-22 and IL-17A; suggesting a critical role for this cytokine triad in establishing host anti-microbial immunity during the acute and effectors phase of infection. In addition, to their known anti-microbial functions; IL-17A and IL-17F reduced the number of intracellular C. jejuni in intestinal epithelia, highlighting a novel aspect of how IL-17 family members may contribute to protective immunity against C. jejuni.

Campylobacter jejuni Lipooligosaccharide Sialylation, Phosphorylation, and Amide/Ester Linkage Modifications Fine-tune Human Toll-like Receptor 4 Activation

Background: Campylobacter jejuni lipooligosaccharide (LOS) is a critical determinant of host innate immunity. Results: Three structural features of the LOS moiety vary significantly between strains and in combination impact monocyte activation. Conclusion: Variation of LOS structure suggests that LOS-TLR4 engagement during C. jejuni infection is strain-specific. Significance: Source and variation of LOS structure among C. jejuni strains may impact host proinflammatory responses. Campylobacter jejuni is a leading cause of acute gastroenteritis. C. jejuni lipooligosaccharide (LOS) is a potent activator of Toll-like receptor (TLR) 4-mediated innate immunity. Structural variations of the LOS have been previously reported in the oligosaccharide (OS) moiety, the disaccharide lipid A (LA) backbone, and the phosphorylation of the LA. Here, we studied LOS structural variation between C. jejuni strains associated with different ecological sources and analyzed their ability to activate TLR4 function. MALDI-TOF MS was performed to characterize structural variation in both the OS and LA among 15 different C. jejuni isolates. Cytokine induction in THP-1 cells and primary monocytes was correlated with LOS structural variation in each strain. Additionally, structural variation was correlated with the source of each strain. OS sialylation, increasing abundance of LA d-glucosamine versus 2,3-diamino-2,3-dideoxy-d-glucose, and phosphorylation status all correlated with TLR4 activation as measured in THP-1 cells and monocytes. Importantly, LOS-induced inflammatory responses were similar to those elicited by live bacteria, highlighting the prominent contribution of the LOS component in driving host immunity. OS sialylation status but not LA structure showed significant association with strains clustering with livestock sources. Our study highlights how variations in three structural components of C. jejuni LOS alter TLR4 activation and consequent monocyte activation.

Pseudaminic Acid on Campylobacter jejuni Flagella Modulates Dendritic Cell IL-10 Expression via Siglec-10 Receptor: A Novel Flagellin-Host Interaction

Introduction. Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. At present the identity of host-pathogen interactions that promote successful bacterial colonisation remain ill defined. Herein, we aimed to investigate C. jejuni-mediated effects on dendritic cell (DC) immunity. Results. We found C. jejuni to be a potent inducer of human and murine DC interleukin 10 (IL-10) in vitro, a cellular event that was MyD88- and p38 MAPK-signalling dependent. Utilizing a series of C. jejuni isogenic mutants we found the major flagellin protein, FlaA, modulated IL-10 expression, an intriguing observation as C. jejuni FlaA is not a TLR5 agonist. Further analysis revealed pseudaminic acid residues on the flagella contributed to DC IL-10 expression. We identified the ability of both viable C. jejuni and purified flagellum to bind to Siglec-10, an immune-modulatory receptor. In vitro infection of Siglec-10 overexpressing cells resulted in increased IL-10 expression in a p38-dependent manner. Detection of Siglec-10 on intestinal CD11c+ CD103+ DCs added further credence to the notion that this novel interaction may contribute to immune outcome during human infection. Conclusions. We propose that unlike the Salmonella Typhimurium flagella-TLR5 driven pro-inflammatory axis, C. jejuni flagella instead promote an anti-inflammatory axis via glycan-Siglec-10 engagement.

Selective Activation of Intracellular Signalling Pathways in Dendritic Cells for Cancer Immunotherapy

One of the major challenges in achieving effective anti-cancer immunotherapy is to counteract immunological tolerance. Most tumor-associated antigens (TAAs) are sensed as self. Hence, naturally occurring tolerance towards them has to be overcome. Fortunately, there is increasing evidence that anti-tumor immune responses occur and play a crucial role in the success of well-established anti-neoplastic therapies such as radiotherapy and chemotherapy. In fact, their effectiveness relies on signalling by pattern recognition receptors such as Toll-like receptors (TLRs). TLR signal transduction involves activation of a few well-known pathways, of which nuclear factor κB (NF-κB) and mitogen activated protein kinases (MAPKs) are possibly the best characterized. Therefore, constitutive activation of these pathways in immune cells can potentially enhance anti-tumor immunity, especially when targeted to professional antigen presenting cells (APCs) such as dendritic cells (DCs). Several strategies have been devised to test this hypothesis, including constitutive activation of TLRs, NF-κB and MAPKs (extracellular-signal regulated kinase (ERK), p38 and c-Jun kinase 1 (JNK1)). Activation of these pathways in mouse and human DCs has differential effects in immunogenicity and in many cases, enhanced antitumor immunity in pre-clinical models, establishing the basis for future clinical applications.

Pancreatic amylase is an environmental signal for regulation of biofilm formation and host interaction in Campylobacter jejuni.

ABSTRACT Campylobacter jejuni is a commensal bacterium in the intestines of animals and birds and a major cause of food-borne gastroenteritis in humans worldwide. Here we show that exposure to pancreatic amylase leads to secretion of an α-dextran by C. jejuni and that a secreted protease, Cj0511, is required. Exposure of C. jejuni to pancreatic amylase promotes biofilm formation in vitro, increases interaction with human epithelial cell lines, increases virulence in the Galleria mellonella infection model, and promotes colonization of the chicken ileum. We also show that exposure to pancreatic amylase protects C. jejuni from stress conditions in vitro, suggesting that the induced α-dextran may be important during transmission between hosts. This is the first evidence that pancreatic amylase functions as an interkingdom signal in an enteric microorganism.

Lentiviral vectors and gene therapy

Lentiviral vectors and gene therapy / , Lentiviral vectors and gene therapy / , کتابخانه دیجیتال جندی شاپور اهواز

Amino acid-based formula affects the gastrointestinal cytokine milieu of children with non-IgE mediated cow’s milk allergy.

Food Allergy (FA) presents a significant health and economic burden in the western world. Children with non-IgE mediated cows milk allergy (CMA) are being increasingly seen in clinic. Diagnosis is largely based on delayed onset of symptoms, primarily affecting the gastrointestinal (GI) mucosa. Treatment involves an elimination diet supplemented with amino acid-based formula (AAF) which in some children results in effective symptom relief. To understand the beneficial effects of AAF at the molecular level, herein we characterized the GI cytokine milieu ex vivo from children with or without AAF in their elimination diets.

Clinical Grade Lentiviral Vectors

Thirty years of extensive research culminated in the year 2000 with the publication of the first clearly successful human gene therapy clinical trial. The trial corrected X-linked severe combined immunodeficiency (SCID-X1) in children using a therapeutic γ-retrovirus vector. Soon afterwards, the results of several other trials were published. More recently, lentiviral vectors have been used for the correction of human β-thalassaemia and adrenoleukodystrophy. In this chapter, we discuss the production of clinical grade retro and lentivectors for their application in human therapy.

Human Gene Therapy with Retrovirus and Lentivirus Vectors

The first human gene therapy clinical trial unsuccessfully took place in the 1970s. Despite extensive research and development in this subject, it was only approximately 10 years ago in 2000, that the results from a completely successful human gene therapy trial were published. Severe combined immunodeficiency X1 was corrected by ex vivo transduction of autologous hematopoietic stem cells with a γ-retrovirus vector encoding the therapeutic gene, followed by retransplantation. Since then, several other clinical trials using retro and lentivectors have followed. In this chapter we will briefly describe and discuss these successful trials for the correction of genetic diseases.