Maladaptive T-Cell Metabolic Fitness in Autoimmune Diseases.

Immune surveillance and adaptive immune responses, involving continuously circulating and tissue-resident T-lymphocytes, provide host defense against infectious agents and possible malignant transformation while avoiding autoimmune tissue damage. Activation, migration, and deployment of T-cells to affected tissue sites are crucial for mounting an adaptive immune response. An effective adaptive immune defense depends on the ability of T-cells to dynamically reprogram their metabolic requirements in response to environmental cues. Inability of the T-cells to adapt to specific metabolic demands may skew cells to become either hyporesponsive (creating immunocompromised conditions) or hyperactive (causing autoimmune tissue destruction). Here, we review maladaptive T-cell metabolic fitness that can cause autoimmune diseases and discuss how T-cell metabolic programs can potentially be modulated to achieve therapeutic benefits.

The Immune Underpinnings of Barrett's-Associated Adenocarcinogenesis: a Retrial of Nefarious Immunologic Co-Conspirators.

There is no doubt that chronic gastroesophageal reflux disease increases the risk of esophageal adenocarcinoma (EAC) by several fold (odds ratio, 6.4; 95% CI, 4.6-9.1), and some relationships between reflux disease-mediated inflammation and oncogenic processes have been explored; however, the precise interconnections between the immune response and genomic instabilities underlying these pathologic processes only now are emerging. Furthermore, the precise cell of origin of the precancerous stages associated with EAC development, Barrett's esophagus, be it cardia resident or embryonic remnant, may shape our interpretation of the likely immune drivers. This review integrates the current collective knowledge of the immunology underlying EAC development and outlines a framework connecting proinflammatory pathways, such as those mediated by interleukin 1ß, tumor necrosis factor α, leukemia inhibitory factor, interleukin 6, signal transduction and activator of transcription 3, nuclear factor-κB, cyclooxygenase-2, and transforming growth factor ß, with oncogenic pathways in the gastroesophageal reflux disease-Barrett's esophagus-EAC cancer sequence. Further defining these immune and molecular railroads may show a map of the routes taken by gastroesophageal cells on their journey toward EAC tumor phylogeny. The selective pressures applied by this immune-induced journey likely impact the phenotype and genotype of the resulting oncogenic destination and further exploration of lesser-defined immune drivers may be useful in future individualized therapies or enhanced selective application of recent immune-driven therapeutics.

Purification of Total RNA from Stomach Tissue Biopsies.

In order to further our understanding of the physiological consequences of Helicobacter pylori infection , analysis of clinical tissue specimens is required. To this end, RNA is frequently isolated from stomach biopsies of H. pylori-infected patients and compared to samples from uninfected controls to monitor gene expression using molecular methods such as reverse-transcription real-time PCR, microarrays, and next-generation sequencing. The successful purification of sufficient quantities of high-quality RNA is essential for accurate and reproducible downstream analysis. This chapter describes the key steps for high-quality RNA purification from human tissue samples, including sample collection and storage, tissue disruption and lysis, RNA purification, and assessment of RNA yield and quality.

Combination Therapy Using Inhalable GapmeR and Recombinant ACE2 for COVID-19.

Here we report our perspective on applying GapmeR technology in combination with recombinant angiotensin-converting enzyme 2 (ACE2) in the treatment of COVID-19 patients. GapmeR is a cell-permeating antisense single-stranded DNA molecule that can be designed to specifically target intracellular severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Once internalized into host cells, such as lung alveolar cells, GapmeR molecules can bind to the viral RNA. This RNA/DNA hybrid will then be degraded by the RNase H enzyme abundantly present in the host cells. GapmeRs can be delivered to COVID-19 patients through inhalation or via nebulization. SARS-CoV-2-targeted GapmeR can also be given to frontline healthcare workers as a prophylactic protection. The recombinant ACE2 protein, the efficacy of which is being evaluated in clinical trials, will bind to the spike (S) glycoprotein of extracellular SARS-CoV-2 and potentially block viral infectivity. We propose that combining inhalable SARS-CoV-2-targeted GapmeRs with recombinant ACE2 could provide a viable and rapidly implementable more effective therapeutic approach for eradicating SARS-CoV-2 and save millions of lives.

High Content Imaging of Barrett's-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions.

BACKGROUND & AIMS: Esophageal adenocarcinoma (EAC) develops from within Barrett's esophagus (BE) concomitant with gastroesophageal reflux disease (GERD). Wound healing processes and cellular transitions, such as epithelial-mesenchymal transitions, may contribute to the development of BE and the eventual migratory escape of metastatic cancer cells. Herein, we attempt to identify the genes underlying esophageal cellular transitions and their potential regulation by the low pH environments observed in GERD and commonly encountered by escaping cancer cells. METHODS: Small interfering RNA library screening and high-content imaging analysis outlined changes in BE high-grade dysplasia (HGD) and EAC cell morphologies after gene silencing. Gene expression microarray data and low pH exposures studies modeling GERD-associated pulses (pH 4.0, 10 min) and tumor microenvironments (pH 6.0, constant) were used. RESULTS: Statistical analysis of small interfering RNA screening data defined 207 genes (Z-score >2.0), in 12 distinct morphologic clusters, whose suppression significantly altered BE-HGD cell morphology. The most significant genes in this list included KIF11, RRM2, NUBP2, P66BETA, DUX1, UBE3A, ITGB8, GAS1, GPS1, and PRC1. Guided by gene expression microarray study data, both pulsatile and constant low pH exposures were observed to suppress the expression of GPS1 and RRM2 in a nonoverlapping temporal manner in both BE-HGD and EAC cells, with no changes observed in squamous esophageal cells. Functional studies uncovered that GPS1 and RRM2 contributed to amoeboid and mesenchymal cellular transitions, respectively, as characterized by differential rates of cell motility, pseudopodia formation, and altered expression of the mesenchymal markers vimentin and E-cadherin. CONCLUSIONS: Collectively, we have shown that low pH microenvironments associated with GERD, and tumor invasive edges, can modulate the expression of genes that triggered esophageal cellular transitions potentially critical to colonization and invasion.

CG-NAP/Kinase Interactions Fine-Tune T Cell Functions.

CG-NAP, also known as AKAP450, is an anchoring/adaptor protein that streamlines signal transduction in various cell types by localizing signaling proteins and enzymes with their substrates. Great efforts are being devoted to elucidating functional roles of this protein and associated macromolecular signaling complex. Increasing understanding of pathways involved in regulating T lymphocytes suggests that CG-NAP can facilitate dynamic interactions between kinases and their substrates and thus fine-tune T cell motility and effector functions. As a result, new binding partners of CG-NAP are continually being uncovered. Here, we review recent advances in CG-NAP research, focusing on its interactions with kinases in T cells with an emphasis on the possible role of this anchoring protein as a target for therapeutic intervention in immune-mediated diseases.

A pilot study of the impact of Vitamin C supplementation with neoadjuvant chemoradiation on regulators of inflammation and carcinogenesis in esophageal cancer patients.

AIMS: Vitamin C plays a role in chemoprevention in cancer treatment, and Vitamin C modulates many regulators of inflammation in in vitro studies. The aim of this study is to assess the effect of Vitamin C supplementation with neoadjuvant chemoradiation in esophageal adenocarcinoma on the nuclear factor-kappa B (NF-κB) and associated cytokines. MATERIALS AND METHODS: A total of 20 patients undergoing multimodal treatment for esophageal adenocarcinoma were randomized to receive Vitamin C (1000 mg/day) orally for 4 weeks or no supplementation. Pre- and post-Vitamin C endoscopic biopsies were used for the study of NF-κB activity and cytokine analysis. RESULTS: NF-κB activity along with cytokines was activated in the cancer tissue pretreatment. Down-regulation in NF-κB activity was observed in 25% of cases, two from the Vitamin C arm posttreatment. There was a significant reduction in cytokines levels in the cancer group, and this effect was more pronounced in the Vitamin C group (P < 0.05). CONCLUSIONS: Vitamin C supplementation had a mild protective effect in modulating of regulators of inflammation and carcinogenesis. Further studies with larger numbers of endpoints are needed to evaluate its effect on modulation of chemoradiation responses.

An Introduction to LFA-1/ICAM-1 Interactions in T-Cell Motility.

The inherent ability of T-cells to migrate is critical for a fully functional immune system, both in normal immune surveillance and for mounting an adaptive immune response. At the same time, inappropriate trafficking of T-cells can be a pathological factor for immune-mediated or chronic inflammatory diseases. T-cell motility is critically dependent on a series of ligand-receptor interactions, a precisely regulated intracellular signaling, an involvement of adaptor proteins, and dynamic remodeling of the cytoskeletal systems. The leukocyte integrin LFA-1 receptor present on T-cells binds to the ligand intercellular adhesion molecule 1 (ICAM-1) and this LFA-1/ICAM-1 contact acts as a trigger for T-cell motility. In this book, we present a collection of methods and protocols that are frequently used by researchers to better understand T-cell motility in health and diseases.

A Laboratory Model to Study T-Cell Motility.

Regulated migration of T-lymphocytes through high endothelial venules and secondary lymphoid organs is necessary for an adaptive immune response. Uncontrolled trafficking of T-cells is implicated in many pathological conditions, including autoimmune disorders, such as psoriasis and inflammatory bowel disease. T-cell migration is regulated mainly by the αLß2 integrin receptor LFA-1, which interacts primarily with its cognate ligand ICAM-1 expressed on the endothelium. This interaction triggers a plethora of downstream signaling pathways, which are not fully understood. Thus, in order to dissect the signal transduction processes at molecular levels and phenotypic changes in migrating T-cells, a laboratory model mimicking T-cell motility is important. Here, we describe a simple and highly reproducible in vitro model to study T-cell migration.

Quantification of T-Cell Migratory Phenotypes Using High-Content Analysis.

The exploration screening of phenotypic changes in motile T-cells within a signaling environment has always been an arduous task due to the sheer population of these microscopic cells. In recent years, High-Content Analysis (HCA) has gained epochal momentum and has allowed for a wider range of quantitative multiplexed cell-based assays in the field of lymphocyte signaling. In this chapter, we consolidate our understanding and describe the technical approach and methodology to quantify T-cell migratory phenotypes using HCA. Optimizations to be adopted to generate high-quality cytological images of motile T-cells and subsequent analysis using HCA are detailed as well.

siRNA Library Screening Identifies a Druggable Immune-Signature Driving Esophageal Adenocarcinoma Cell Growth.

BACKGROUND & AIMS: Effective therapeutic approaches are urgently required to tackle the alarmingly poor survival outcomes in esophageal adenocarcinoma (EAC) patients. EAC originates from within the intestinal-type metaplasia, Barrett's esophagus, a condition arising on a background of gastroesophageal reflux disease and associated inflammation. METHODS: This study used a druggable genome small interfering RNA (siRNA) screening library of 6022 siRNAs in conjunction with bioinformatics platforms, genomic studies of EAC tissues, somatic variation data of EAC from The Cancer Genome Atlas data of EAC, and pathologic and functional studies to define novel EAC-associated, and targetable, immune factors. RESULTS: By using a druggable genome library we defined genes that sustain EAC cell growth, which included an unexpected immunologic signature. Integrating Cancer Genome Atlas data with druggable siRNA targets showed a striking concordance and an EAC-specific gene amplification event associated with 7 druggable targets co-encoded at Chr6p21.1. Over-representation of immune pathway-associated genes supporting EAC cell growth included leukemia inhibitory factor, complement component 1, q subcomponent A chain (C1QA), and triggering receptor expressed on myeloid cells 2 (TREM2), which were validated further as targets sharing downstream signaling pathways through genomic and pathologic studies. Finally, targeting the triggering receptor expressed on myeloid cells 2-, C1q-, and leukemia inhibitory factor-activated signaling pathways (TYROBP-spleen tyrosine kinase and JAK-STAT3) with spleen tyrosine kinase and Janus-activated kinase inhibitor fostamatinib R788 triggered EAC cell death, growth arrest, and reduced tumor burden in NOD scid gamma mice. CONCLUSIONS: These data highlight a subset of genes co-identified through siRNA targeting and genomic studies of expression and somatic variation, specifically highlighting the contribution that immune-related factors play in support of EAC development and suggesting their suitability as targets in the treatment of EAC.

Evidence of CD4+ T cell-mediated immune pressure on the Hepatitis C virus genome.

Hepatitis C virus (HCV)-specific T cell responses are critical for immune control of infection. Viral adaptation to these responses, via mutations within regions of the virus targeted by CD8+ T cells, is associated with viral persistence. However, identifying viral adaptation to HCV-specific CD4+ T cell responses has been difficult although key to understanding anti-HCV immunity. In this context, HCV sequence and host genotype from a single source HCV genotype 1B cohort (n = 63) were analyzed to identify viral changes associated with specific human leucocyte antigen (HLA) class II alleles, as these variable host molecules determine the set of viral peptides presented to CD4+ T cells. Eight sites across the HCV genome were associated with HLA class II alleles implicated in infection outcome in this cohort (p ≤ 0.01; Fisher's exact test). We extended this analysis to chronic HCV infection (n = 351) for the common genotypes 1A and 3A. Variation at 38 sites across the HCV genome were associated with specific HLA class II alleles with no overlap between genotypes, suggestive of genotype-specific T cell targets, which has important implications for vaccine design. Here we show evidence of HCV adaptation to HLA class II-restricted CD4+ T cell pressure across the HCV genome in chronic HCV infection without a priori knowledge of CD4+ T cell epitopes.

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.

Epidemiology of Sapovirus Infections in a Birth Cohort in Peru.

Background: Sapovirus is one of the primary viral causes of acute gastroenteritis (AGE), especially where rotavirus vaccination has been implemented. The characteristics and impact of natural infection at the community level, however, have not been well documented. Methods: Stool samples were analyzed from 100 children randomly selected from a community-based birth cohort study in Peru. All diarrheal and 1 nondiarrheal stools collected trimonthly from children up to age 2 years (n = 1669) were tested for sapovirus detection. Viral shedding duration was determined by testing additional weekly samples (n = 440) collected before and after a sapovirus-positive sample. Results: The incidence of sapovirus infection in the first and second years of life was 4.3 and 11.1 per 100 child-months, respectively. By age 2 years, 82% of children had at least 1 sapovirus infection, and 64% had at least 1 sapovirus-associated diarrhea episode. The median shedding period was 18.5 days. In 112 of 175 infections, 14 genotypes from 4 genogroups (GI, GII, GIV, and GV) were determined. Among genogroups, GI were more frequently found in symptomatic infections than in asymptomatic infections (odds ratio, 3.1; 95% confidence interval, 1.3-7.4). Fifty-nine children had serial sapovirus infections, but only 3 had repeated infection of the same genotype. Conclusions: Sapovirus was frequently detected in children with AGE at the community level during the first 2 years of life. Serial sapovirus infections by multiple genotypes in a child suggest genotype-specific immunity from each infection, which needs to be taken into account for vaccine development.

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 Barrett's 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 Barrett's 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.

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

The αLß2 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-ß 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.

Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response.

BACKGROUND: Clostridium 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. RESULTS: We 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. CONCLUSIONS: These 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.

International, evidence-based consensus diagnostic criteria for HHV-8-negative/idiopathic multicentric Castleman disease.

Human herpesvirus-8 (HHV-8)-negative, idiopathic multicentric Castleman disease (iMCD) is a rare and life-threatening disorder involving systemic inflammatory symptoms, polyclonal lymphoproliferation, cytopenias, and multiple organ system dysfunction caused by a cytokine storm often including interleukin-6. iMCD accounts for one third to one half of all cases of MCD and can occur in individuals of any age. Accurate diagnosis is challenging, because no standard diagnostic criteria or diagnostic biomarkers currently exist, and there is significant overlap with malignant, autoimmune, and infectious disorders. An international working group comprising 34 pediatric and adult pathology and clinical experts in iMCD and related disorders from 8 countries, including 2 physicians that are also iMCD patients, was convened to establish iMCD diagnostic criteria. The working group reviewed data from 244 cases, met twice, and refined criteria over 15 months (June 2015 to September 2016). The proposed consensus criteria require both Major Criteria (characteristic lymph node histopathology and multicentric lymphadenopathy), at least 2 of 11 Minor Criteria with at least 1 laboratory abnormality, and exclusion of infectious, malignant, and autoimmune disorders that can mimic iMCD. Characteristic histopathologic features may include a constellation of regressed or hyperplastic germinal centers, follicular dendritic cell prominence, hypervascularization, and polytypic plasmacytosis. Laboratory and clinical Minor Criteria include elevated C-reactive protein or erythrocyte sedimentation rate, anemia, thrombocytopenia or thrombocytosis, hypoalbuminemia, renal dysfunction or proteinuria, polyclonal hypergammaglobulinemia, constitutional symptoms, hepatosplenomegaly, effusions or edema, eruptive cherry hemangiomatosis or violaceous papules, and lymphocytic interstitial pneumonitis. iMCD consensus diagnostic criteria will facilitate consistent diagnosis, appropriate treatment, and collaborative research.