Dermot Kelleher

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

Background & Aims Barretts esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barretts and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. Methods We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. Results We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09–1.18; P = 1.8 × 10−11) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86–0.93; P = 7.5 × 10−9). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87–0.93; P = 3.72 × 10−9). Conclusions We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.

Phosphorylation of Rab5a protein by protein kinase Cϵ is crucial for T-cell migration

Background: Rab5a GTPase plays important roles in intracellular transport and cell signaling. Results: T-cell stimulation through the integrin LFA-1 or the chemokine receptor CXCR4 induces PKCϵ-dependent phosphorylation of Rab5a at Thr-7, which is crucial for cytoskeleton remodeling and cell migration. Conclusion: PKCϵ-Rab5a-Rac1 axis regulates T-cell motility. Significance: The study provides novel insights into the role of Rab5a in the adaptive immune response. Rab GTPases control membrane traffic and receptor-mediated endocytosis. Within this context, Rab5a plays an important role in the spatial regulation of intracellular transport and signal transduction processes. Here, we report a previously uncharacterized role for Rab5a in the regulation of T-cell motility. We show that Rab5a physically associates with protein kinase Cϵ (PKCϵ) in migrating T-cells. After stimulation of T-cells through the integrin LFA-1 or the chemokine receptor CXCR4, Rab5a is phosphorylated on an N-terminal Thr-7 site by PKCϵ. Both Rab5a and PKCϵ dynamically interact at the centrosomal region of migrating cells, and PKCϵ-mediated phosphorylation on Thr-7 regulates Rab5a trafficking to the cell leading edge. Furthermore, we demonstrate that Rab5a Thr-7 phosphorylation is functionally necessary for Rac1 activation, actin rearrangement, and T-cell motility. We present a novel mechanism by which a PKCϵ-Rab5a-Rac1 axis regulates cytoskeleton remodeling and T-cell migration, both of which are central for the adaptive immune response.

The collaborative network approach: a new framework to accelerate Castleman's disease and other rare disease research

Despite technological advances and substantial investments of time and funding, many challenges exist for rare disease research and drug development. Basic disease mechanisms are often poorly understood, selection of suitable research participants can be diffi cult, and endpoints for clinical trial registration might not be established. Progress is further impeded by limited collaboration and data coordination, misaligned incentives, funding decisions made in isolation from community consensus, and inter-institutional barriers to tissue sharing. Although frequently used, off -label treatments are infrequently tracked, and thus valuable opportunities to build on observations are lost. One coauthor (DCF) comments, “Despite being a physician, I didn’t fully understand the barriers that slowed down life-saving progress until I was dying from a rare illness. I received my last rites in 2010 while battling idiopathic multicentric Castleman’s disease and found that [the disease] had no Food and Drug Administration [FDA]-approved therapies, a poorly understood model of pathogenesis, and a 65% 5-year survival rate”. After surviving three life-threatening episodes, DCF began to assess how non-profi t research funding was allocated for rare diseases, for which 95% of conditions do not have an FDA-approved therapy. Disease research organisations (DROs) make important contributions by funding research. However, the framework through which many (but not all) DROs traditionally distribute funding can present challenges (fi gure). DROs typically fundraise fi rst and then invite researchers to apply through a request for proposals (RFP) to use the funding for their specifi c purposes. In parallel, these organisations often provide patients with supportive resources and facilitate referrals to experts, who collect and store clinical data and biomaterials at their respective institutions. This model can in some cases result in organisations funding studies targeted at questions proposed by a small selection of researchers with requisite tissue samples rather than the studies that may have the highest impact on the fi eld, which might require collaboration and sample sharing. A more effi cient, collaborative, and consensus-driven framework than exists at present is needed to fully harness the opportunities aff orded by technological advances for the approximately 7000 rare diseases and 350 million individuals affl icted globally. One such neglected rare condition is Castleman’s disease, which describes a heterogeneous group of lymphoproliferative disorders straddling the fi elds of immunology, oncology, and virology. Castleman’s disease ranges from a single region of enlarged lymph nodes (unicentric Castleman’s disease) to multicentric lymphadenopathy with systemic infl ammation and multiple organ system dysfunction (multicentric Castleman’s disease) caused by immune activation and proinfl ammatory hypercytokinaemia, often including interleukin 6. Multicentric Castleman’s disease is either caused by human herpesvirus 8 (HHV8) infection, which is called HHV8-associated multicentric Castleman’s disease, or is idiopathic in HHV8-negative patients and called idiopathic multicentric Castleman’s disease. The absence of a unique international classifi cation of disease (ICD) code for Castleman’s disease has made epidemiological studies diffi cult. The estimated incidence of all forms of Castleman’s disease is 6500–7700 individuals of all ages every year in the USA or about 2·2 per 100 000.

Adaptor regulation of LFA-1 signaling in T lymphocyte migration : potential druggable targets for immunotherapies?

The integrin lymphocyte function associated antigen‐1 (LFA‐1) plays a key role in leukocyte trafficking and in adaptive immune responses through interactions with adhesive ligands, such as ICAM‐1. Specific blockade of these interactions has validated LFA‐1 as a therapeutic target in many chronic inflammatory diseases, however LFA‐1 antagonists have not been clinically successful due to the development of a general immunosuppression, causing fatal side effects. Growing evidence has now established that LFA‐1 mediates an array of intracellular signaling pathways by triggering a number of downstream molecules. In this context, a class of multimodular domain‐containing proteins capable of recruiting two or more effector molecules, collectively known as “adaptor proteins,” has emerged as important mediators in LFA‐1 signal transduction. Here, we provide an overview of the adaptor proteins involved in the intracellular signaling cascades by which LFA‐1 regulates T‐cell motility and immune responses. The complexity of the LFA‐1‐associated signaling delineated in this review suggests that it may be an important and challenging focus for future research, enabling the identification of “tunable” targets for the development of immunotherapies.

LFA-1/ICAM-1 Ligation in Human T Cells Promotes Th1 Polarization through a GSK3β Signaling–Dependent Notch Pathway

In this study, we report that the integrin LFA-1 cross-linking with its ligand ICAM-1 in human PBMCs or CD4+ T cells promotes Th1 polarization by upregulating IFN-γ secretion and T-bet expression. LFA-1 stimulation in PBMCs, CD4+ T cells, or the T cell line HuT78 activates the Notch pathway by nuclear translocation of cleaved Notch1 intracellular domain (NICD) and upregulation of target molecules Hey1 and Hes1. Blocking LFA-1 by a neutralizing Ab or specific inhibition of Notch1 by a γ-secretase inhibitor substantially inhibits LFA-1/ICAM-1–mediated activation of Notch signaling. We further demonstrate that the Notch pathway activation is dependent on LFA-1/ICAM-1–induced inactivation of glycogen synthase kinase 3β (GSK3β), which is mediated via Akt and ERK. Furthermore, in silico analysis in combination with coimmunoprecipitation assays show an interaction between NICD and GSK3β. Thus, there exists a molecular cross-talk between LFA-1 and Notch1 through the Akt/ERK–GSK3β signaling axis that ultimately enhances T cell differentiation toward Th1. Although clinical use of LFA-1 antagonists is limited by toxicity related to immunosuppression, these findings support the concept that Notch inhibitors could be attractive for prevention or treatment of Th1-related immunologic disorders and have implications at the level of local inflammatory responses.

Not Just an Adhesion Molecule: LFA-1 Contact Tunes the T Lymphocyte Program

The αLβ2 integrin LFA-1 is known to play a key role in T lymphocyte migration, which is necessary to mount a local immune response, and is also the main driver of autoimmune diseases. This migration-triggering signaling process in T cells is tightly regulated to permit an immune response that is appropriate to the local trigger, as well as to prevent deleterious tissue-damaging bystander effects. Emerging evidence shows that, in addition to prompting a diverse range of downstream signaling cascades, LFA-1 stimulation in T lymphocytes modulates gene-transcription programs, including genetic signatures of TGF-β and Notch pathways, with multifactorial biological outcomes. This review highlights recent findings and discusses molecular mechanisms by which LFA-1 signaling influence T lymphocyte differentiation into the effector subsets Th1, Th17, and induced regulatory T cells. We argue that LFA-1 contact with a cognate ligand, such as ICAM-1, independent of the immune synapse activates a late divergence in T cells’ effector phenotypes, hence fine-tuning their functioning.

Centrosome- and Golgi-Localized Protein Kinase N-Associated Protein Serves As a Docking Platform for Protein Kinase A Signaling and Microtubule Nucleation in Migrating T-Cells

Centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP), also known as AKAP450, is a cytosolic scaffolding protein involved in the targeted positioning of multiple signaling molecules, which are critical for cellular functioning. Here, we show that CG-NAP is predominantly expressed in human primary T-lymphocytes, localizes in close proximity (<0.2 μm) with centrosomal and Golgi structures and serves as a docking platform for Protein Kinase A (PKA). GapmeR-mediated knockdown of CG-NAP inhibits LFA-1-induced T-cell migration and impairs T-cell chemotaxis toward the chemokine SDF-1α. Depletion of CG-NAP dislocates PKARIIα, disrupts centrosomal and non-centrosomal microtubule nucleation, causes Golgi fragmentation, and impedes α-tubulin tyrosination and acetylation, which are important for microtubule dynamics and stability in migrating T-cells. Furthermore, we show that CG-NAP coordinates PKA-mediated phosphorylation of pericentrin and dynein in T-cells. Overall, our findings provide critical insights into the roles of CG-NAP in regulating cytoskeletal architecture and T-cell migration.

Deoxycholic acid promotes development of gastroesophageal reflux disease and Barrett's oesophagus by modulating integrin‐αv trafficking

The fundamental mechanisms underlying erosive oesophagitis and subsequent development of Barretts oesophagus (BO) are poorly understood. Here, we investigated the contribution of specific components of the gastric refluxate on adhesion molecules involved in epithelial barrier maintenance. Cell line models of squamous epithelium (HET‐1A) and BO (QH) were used to examine the effects of bile acids on cell adhesion to extracellular matrix proteins (Collagen, laminin, vitronectin, fibronectin) and expression of integrin ligands (α3, α4, α5, α6 and αν). Experimental findings were validated in human explant oesophageal biopsies, a rat model of gastroesophageal reflux disease (GORD) and in patient tissue microarrays. The bile acid deoxycholic acid (DCA) specifically reduced adhesion of HET‐1A cells to vitronectin and reduced cell‐surface expression of integrin‐αν via effects on endocytic recycling processes. Increased expression of integrin‐αv was observed in ulcerated tissue in a rat model of GORD and in oesophagitis and Barretts intestinal metaplasia patient tissue compared to normal squamous epithelium. Increased expression of integrin‐αν was observed in QH BO cells compared to HET‐1A cells. QH cells were resistant to DCA‐mediated loss of adhesion and reduction in cell‐surface expression of integrin‐αν. We demonstrated that a specific component of the gastric refluxate, DCA, affects the epithelial barrier through modulation of integrin αν expression, providing a novel mechanism for bile acid‐mediated erosion of oesophageal squamous epithelium and promotion of BO. Strategies aimed at preventing bile acid‐mediated erosion should be considered in the clinical management of patients with GORD.

RNAi Screening with Self-Delivering, Synthetic siRNAs for Identification of Genes That Regulate Primary Human T Cell Migration

Screening of RNA interference (RNAi) libraries in primary T cells is labor-intensive and technically challenging because these cells are hard to transfect. Chemically modified, self-delivering small interfering RNAs (siRNAs) offer a solution to this problem, because they enter hard-to-transfect cell types without needing a delivery reagent and are available in library format for RNAi screening. In this study, we have screened a library of chemically modified, self-delivering siRNAs targeting the expression of 72 distinct genes in conjunction with an image-based high-content-analysis platform as a proof-of-principle strategy to identify genes involved in lymphocyte function-associated antigen-1 (LFA-1)-mediated migration in primary human T cells. Our library-screening strategy identified the small GTPase RhoA as being crucial for T cell polarization and migration in response to LFA-1 stimulation and other migratory ligands. We also demonstrate that multiple downstream assays can be performed within an individual RNAi screen and have used the remainder of the cells for additional assays, including cell viability and adhesion to ICAM-1 (the physiological ligand for LFA-1) in the absence or presence of the chemokine SDF-1α. This study therefore demonstrates the ease and benefits of conducting siRNA library screens in primary human T cells using self-delivering, chemically modified siRNAs, and it emphasizes the feasibility and potential of this approach for elucidating the signaling pathways that regulate T cell function.

Surface Layer Proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response

BackgroundClostridium difficile is a nosocomial pathogen prevalent in hospitals worldwide and increasingly common in the community. Sequence differences have been shown to be present in the Surface Layer Proteins (SLPs) from different C. difficile ribotypes (RT) however whether these differences influence severity of infection is still not clear.ResultsWe used a molecular evolutionary approach to analyse SLPs from twenty-six C. difficile RTs representing different slpA sequences. We demonstrate that SLPs from RT 027 and 078 exhibit evidence of positive selection (PS). We compared the effect of these SLPs to those purified from RT 001 and 014, which did not exhibit PS, and demonstrate that the presence of sites under positive selection correlates with ability to activate macrophages. SLPs from RTs 027 and 078 induced a more potent response in macrophages, with increased levels of IL-6, IL-12p40, IL-10, MIP-1α, MIP-2 production relative to RT 001 and 014. Furthermore, RTs 027 and 078 induced higher expression of CD40, CD80 and MHC II on macrophages with decreased ability to phagocytose relative to LPS.ConclusionsThese results tightly link sequence differences in C. difficile SLPs to disease susceptibility and severity, and suggest that positively selected sites in the SLPs may play a role in driving the emergence of hyper-virulent strains.