RIG-I and Other RNA Sensors in Antiviral Immunity.

Pattern recognition receptors (PRRs) survey intra- and extracellular spaces for pathogen-associated molecular patterns (PAMPs) within microbial products of infection. Recognition and binding to cognate PAMP ligand by specific PRRs initiates signaling cascades that culminate in a coordinated intracellular innate immune response designed to control infection. In particular, our immune system has evolved specialized PRRs to discriminate viral nucleic acid from host. These are critical sensors of viral RNA to trigger innate immunity in the vertebrate host. Different families of PRRs of virus infection have been defined and reveal a diversity of PAMP specificity for wide viral pathogen coverage to recognize and extinguish virus infection. In this review, we discuss recent insights in pathogen recognition by the RIG-I-like receptors, related RNA helicases, Toll-like receptors, and other RNA sensor PRRs, to present emerging themes in innate immune signaling during virus infection.

SnapShot: Interferon Signaling.

Interferons (IFNs) are crucial cytokines of antimicrobial, antitumor, and immunomodulatory activity. The three types of IFN (I, II, and III) are classified by their receptor specificity and sequence homology. IFNs are produced and secreted by cells in response to specific stimuli. Here, we review the subsequent IFN signaling events occurring through unique receptors leading to regulation of gene expression for modulation of innate and adaptive immunity. To view this SnapShot, open or download the PDF.

Promotion and Detection of Cell-Cell Interactions through a Bioorthogonal Approach.

Manipulation of cell-cell interactions via cell surface modification is crucial in tissue engineering and cell-based therapy. To be able to monitor intercellular interactions, it can also provide useful information for understanding how the cells interact and communicate. We report herein a facile bioorthogonal strategy to promote and monitor cell-cell interactions. It involves the use of a maleimide-appended tetrazine-caged boron dipyrromethene (BODIPY)-based fluorescent probe and a maleimide-substituted bicyclo[6.1.0]non-4-yne (BCN) to modify the membrane of macrophage (RAW 264.7) and cancer (HT29, HeLa, and A431) cells, respectively, via maleimide-thiol conjugation. After modification, the two kinds of cells interact strongly through inverse electron-demand Diels-Alder reaction of the surface tetrazine and BCN moieties. The coupling also disrupts the tetrazine quenching unit, restoring the fluorescence emission of the BODIPY core on the cell-cell interface, and promotes phagocytosis. Hence, this approach can promote and facilitate the detection of intercellular interactions, rendering it potentially useful for macrophage-based immunotherapy.

Inducing Immunogenic Cancer Cell Death through Oxygen-Economized Photodynamic Therapy with Nitric Oxide-Releasing Photosensitizers.

Photodynamic therapy (PDT) utilizes reactive oxygen species (ROS) for eradication of cancer cells. Its effectiveness is governed by the oxygen content, which is scarce in the hypoxic tumor microenvironment. We report herein two zinc(II) phthalocyanines substituted with two or four nitric oxide (NO)-releasing moieties, namely ZnPc-2NO and ZnPc-4NO, which can suppress the mitochondrial respiration, thereby sparing more intracellular oxygen for PDT. Using HT29 human colorectal adenocarcinoma cells and A549 human lung carcinoma cells, we have demonstrated that both conjugates release NO upon interaction with the intracellular glutathione, which can reduce the cellular oxygen consumption rate and adenosine triphosphate generation and alter the mitochondrial membrane potential. They can also relieve the hypoxic status of cancer cells and decrease the expression of hypoxia-inducible factor protein HIF-1α. Upon light irradiation, both conjugates can generate ROS and induce cytotoxicity even under a hypoxic condition, overcoming the oxygen-dependent nature of PDT. Interestingly, the photodynamic action of ZnPc-2NO elicits the release of damage-associated molecular patterns, inducing the maturation of dendritic cells and triggering an antitumor immune response. The immunogenic cell death caused by this oxygen-economized PDT has been demonstrated through a series of in vitro and in vivo experiments.

Site-Selective Lysine Acetylation of Human Immunoglobulin G for Immunoliposomes and Bispecific Antibody Complexes.

Site-selective acetylation of a single lysine residue in a protein that reaches a lysine acetyltransferase's accuracy, precision, and reliability is challenging. Here, we report a peptide-guided, proximity-driven group transfer reaction that acetylates a single lysine residue, Lys 248, of the fragment crystallizable region (Fc region) in the heavy chain of the human Immunoglobulin G (IgG). An Fc-interacting peptide bound with the Fc domain and positioned a phenolic ester close to Lys 248, which induced a nucleophilic reaction and resulted in the transfer of an acetyl group to Lys 248. The acetylation reaction proceeded to a decent yield under the physiological condition without the need for deglycosylation, unnatural amino acids, or catalysts. Along with acetylation, functional moieties such as azide, alkyne, fluorescent molecules, or biotin could also be site-selectively installed on Lys 248, allowing IgG's further derivatization. We then synthesized an antibody-lipid conjugate and constructed antibody-conjugated liposomes (immunoliposomes), targeting HER2-positive (HER2+) cancer cells. We also built a bispecific antibody complex (bsAbC) covalently linking an anti-HER2 antibody and an anti-CD3 antibody. The bsAbC showed in vitro effector-cell-mediated cytotoxicity at nanomolar concentrations. Compared with bispecific antibodies (bsAbs), bsAbCs are constructed based on native IgGs and contain two antigen-binding sites to each antigen, twice that of bsAbs. Altogether, this work reports a method of site-selective acetylation of native antibodies, highlights a facile way of site-selective IgG functionalization, and underscores the potential of bsAbCs in cancer immunotherapy.

Open hip reduction using a novel transarticular suture stabilisation technique in 24 dogs: a retrospective study of technique, outcome and complications.

CASE HISTORY: Medical records of a single private practice (Illinois, USA) were retrospectively reviewed to identify dogs (n = 24) that had an open hip reduction with a transarticular suture stabilisation technique after presenting with a traumatic coxofemoral luxation between April 2003 and December 2018. CLINICAL FINDINGS: Dogs that met the inclusion criteria were of various breeds with a median body weight of 18.1 (min 4.2, max 54.5) kg and mean age at presentation of 6.5 (min 1, max 11) years. The surgical technique, short-term outcome and complications were extracted from the medical records. Long-term (>2 years) follow-up data was obtained by a telephone interview with each owner. TREATMENT AND OUTCOME: All dogs underwent open hip reduction using a novel transarticular suture stabilisation technique. The outcome was reported by owners to be excellent in 18/24 (75%) dogs with full return of limb function. Sixty-six percent (16/24) of owners reported that no lameness was observed 2 months after surgery. No minor complications were noted in this study. The hips of 6/24 (25%) dogs reluxated after surgery (defined as a major complication), which required femoral head and neck excision surgery. CLINICAL RELEVANCE: Open coxofemoral joint reduction using a novel transarticular suture technique is a viable surgical option to consider in dogs that present with a traumatic coxofemoral luxation.

Differential and Overlapping Immune Programs Regulated by IRF3 and IRF5 in Plasmacytoid Dendritic Cells.

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5, an immune-regulatory transcription factor. We show that the protein kinases IKKα, IKKß, IKKε, and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization, thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets, we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs, linking IRF5 with immune regulatory and proinflammatory gene expression. Thus, TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome.

Observations of Multiple Nuclear Reaction Histories and Fuel-Ion Species Dynamics in Shock-Driven Inertial Confinement Fusion Implosions.

Fuel-ion species dynamics in hydrodynamiclike shock-driven DT^{3}He-filled inertial confinement fusion implosion is quantitatively assessed for the first time using simultaneously measured D^{3}He and DT reaction histories. These reaction histories are measured with the particle x-ray temporal diagnostic, which captures the relative timing between different nuclear burns with unprecedented precision (∼10 ps). The observed 50±10 ps earlier D^{3}He reaction history timing (relative to DT) cannot be explained by average-ion hydrodynamic simulations and is attributed to fuel-ion species separation between the D, T, and ^{3}He ions during shock convergence and rebound. At the onset of the shock burn, inferred ^{3}He/T fuel ratio in the burn region using the measured reaction histories is much higher as compared to the initial gas-filled ratio. As T and ^{3}He have the same mass but different charge, these results indicate that the charge-to-mass ratio plays an important role in driving fuel-ion species separation during strong shock propagation even for these hydrodynamiclike plasmas.

Modeling the effects of medial olivocochlear efferent stimulation at the level of the inferior colliculus.

Various studies on medial olivocochlear (MOC) efferents have implicated it in multiple roles in the auditory system (e.g., dynamic range adaptation, masking reduction, and selective attention). This study presents a systematic simulation of inferior colliculus (IC) responses with and without electrical stimulation of the MOC. Phenomenological models of the responses of auditory nerve (AN) fibers and IC neurons were used to this end. The simulated responses were highly consistent with physiological data (replicated 3 of the 4 known rate-level responses all MOC effects-shifts, high stimulus level reduction and enhancement). Complex MOC efferent effects which were previously thought to require integration from different characteristic frequency (CF) neurons were simulated using the same frequency inhibition excitation circuitry. MOC-induced enhancing effects were found only in neurons with a CF range from 750 Hz to 2 kHz. This limited effect is indicative of the role of MOC activation on the AN responses at the stimulus offset.

Piloting a partially self-financed mode of human immunodeficiency virus pre-exposure prophylaxis delivery for men who have sex with men in Hong Kong.

INTRODUCTION: Pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate (TDF) 300 mg/emtricitabine (FTC) 200 mg is a proven strategy for preventing human immunodeficiency virus (HIV) transmission in men who have sex with men (MSM). This study aimed to test the feasibility and acceptability of PrEP delivered at a pilot clinic for MSM in Hong Kong, where PrEP service is currently unavailable. METHODS: Partially self-financed PrEP was provided to HIV-negative adult MSM with high behavioural risk of HIV transmission after excluding hepatitis B infection and renal insufficiency. Participants received daily TDF/FTC for 30 weeks at 13.3% of the drug cost. Adherence and behaviours were monitored through questionnaires while creatinine and HIV/STI (sexually transmitted infection) incidence were monitored with point-of-care and laboratory tests. Preference for continuing with PrEP was evaluated at the end of the prescription period. RESULTS: Seventy-one PrEP-naïve MSM were included in the study, of whom 57 (80%) were retained at the end of 28 weeks. Satisfactory adherence and self-limiting adverse events were reported, while none of the participants contracted HIV. Risk compensation was observed, with an STI incidence of 3.17 per 100 person-years. At the end of the prescription period, a majority (89%) indicated interest in continuing with PrEP. Preference for PrEP was associated with age ≥28 years and peer influence (P=0.04), while stigma was a concern. Price was a deterrent to self-financed PrEP, and only half (51%) considered a monthly cost of ≤HK$500 (US$1=HK$7.8) as reasonable. CONCLUSIONS: A partially self-financed mode of PrEP delivery is feasible with good retention in MSM in Hong Kong.

IL-17 deficiency attenuates allograft injury and prolongs survival in a murine model of fully MHC-mismatched renal allograft transplantation.

IL-17 is a pro-inflammatory cytokine implicated in the pathogenesis of inflammatory and autoimmune diseases. However the role of IL-17 in renal allograft rejection has not been fully explored. Here, we investigate the impact of IL-17 in a fully MHC-mismatched, life-sustaining, murine model of kidney allograft rejection using IL-17 deficient donors and recipients (IL-17(-/-) allografts). IL-17(-/-) allografts exhibited prolonged survival which was associated with reduced expression of the Th1 cytokine IFN-γ and histological attenuation of acute and chronic allograft rejection, as compared to wild-type allograft recipients. Results were confirmed in WT allograft recipients treated with an IL-17 blocking antibody. Subsequent experiments using either donors or recipients deficient in IL-17 showed a trend towards prolongation of survival only when recipients were IL-17(-/-) . Administration of a depleting anti-CD25 antibody to IL-17(-/-) recipients abrogated the survival advantage conferred by IL-17 deficiency, suggesting the involvement of a CD4(+) CD25(+) T cell regulatory mechanism. Therefore, IL-17 deficiency or neutralization was protective against the development of kidney allograft rejection, which may be mediated by impairment of Th1 responses and/or enhanced protection by Tregs.

Pulmonary artery sarcoma diagnosed by endobronchial ultrasound-guided transbronchial needle aspiration.

Pulmonary artery sarcoma is a rare disease with poor prognosis that has not been reported in Hong Kong. Its clinical and radiological presentation frequently mimics pulmonary embolism. Diagnosis is usually delayed until surgery, which is the treatment option that provides the best survival. Endobronchial ultrasound-guided transbronchial needle aspiration is an effective non-surgical technique for lymph node staging of lung cancer and diagnosis of mediastinal lesions via bronchoscopy. Here we discuss a case of pulmonary artery sarcoma diagnosed by this method, the second one in the literature, which serves to illustrate its potential use for early and minimally invasive diagnosis of the condition. Although such aspiration is a safe procedure, tissue sampling of extravascular extensions is advisable wherever possible.

TLR4 sensitizes plasmacytoid dendritic cells for antiviral response against SARS-CoV-2 coronavirus.

Plasmacytoid dendritic cells are a rare subset of dendritic cells that exhibit antiviral functions in response to toll-like receptor 7/8 stimulations. Alternative toll-like receptors such as TLR4 have been known to be active in plasmacytoid dendritic cells for immune regulatory functions. However, it is unclear whether these toll-like receptors differentially activate plasmacytoid dendritic cells as compared with canonical toll-like receptor 7/8 stimulation. Here, we assessed alternative plasmacytoid dendritic cell activation states mediated by toll-like receptors other than endosomal toll-like receptors via the RNA sequencing approach. We found that toll-like receptor 4 stimulation induced a high degree of similarity in gene expression pattern to toll-like receptor 7/8 stimulation in plasmacytoid dendritic cells. Despite high resemblance to toll-like receptor 7/8, we discovered unique genes that were activated under toll-like receptor 4 activation only, as well as genes that were induced at a higher magnitude in comparison to toll-like receptor 7/8 activation. In comparison between toll-like receptor 4-activated plasmacytoid dendritic cells and conventional dendritic cells, we revealed that plasmacytoid dendritic cells and conventional dendritic cells expressed distinct gene sets, whereby conventional dendritic cells mostly favored antigen presentation functions for adaptive immune response regulation while plasmacytoid dendritic cells leaned toward immune response against infectious diseases. Last, we determined that toll-like receptor 4 activation sensitized plasmacytoid dendritic cells against SARS-CoV-2 (COVID-19) single-stranded RNA by enhancing antiviral-related responses and type I interferon production. These findings provided greater insights into the toll-like receptor 4 activation state in plasmacytoid dendritic cells, which can be beneficial for alternative therapeutic interventions involving plasmacytoid dendritic cells for various diseases.

An Immunological Perspective of Circulating Tumor Cells as Diagnostic Biomarkers and Therapeutic Targets.

Immune modulation is a hallmark of cancer. Cancer-immune interaction shapes the course of disease progression at every step of tumorigenesis, including metastasis, of which circulating tumor cells (CTCs) are regarded as an indicator. These CTCs are a heterogeneous population of tumor cells that have disseminated from the tumor into circulation. They have been increasingly studied in recent years due to their importance in diagnosis, prognosis, and monitoring of treatment response. Ample evidence demonstrates that CTCs interact with immune cells in circulation, where they must evade immune surveillance or modulate immune response. The interaction between CTCs and the immune system is emerging as a critical point by which CTCs facilitate metastatic progression. Understanding the complex crosstalk between the two may provide a basis for devising new diagnostic and treatment strategies. In this review, we will discuss the current understanding of CTCs and the complex immune-CTC interactions. We also present novel options in clinical interventions, targeting the immune-CTC interfaces, and provide some suggestions on future research directions.

Identification of AML1-ETO modulators by chemical genomics.

Somatic rearrangements of transcription factors are common abnormalities in the acute leukemias. With rare exception, however, the resultant protein products have remained largely intractable as pharmacologic targets. One example is AML1-ETO, the most common translocation reported in acute myeloid leukemia (AML). To identify AML1-ETO modulators, we screened a small molecule library using a chemical genomic approach. Gene expression signatures were used as surrogates for the expression versus loss of the translocation in AML1-ETO-expressing cells. The top classes of compounds that scored in this screen were corticosteroids and dihydrofolate reductase (DHFR) inhibitors. In addition to modulating the AML1-ETO signature, both classes induced evidence of differentiation, dramatically inhibited cell viability, and ultimately induced apoptosis via on-target activity. Furthermore, AML1-ETO-expressing cell lines were exquisitely sensitive to the effects of corticosteroids on cellular viability compared with nonexpressers. The corticosteroids diminished AML1-ETO protein in AML cells in a proteasome- and glucocorticoid receptor-dependent manner. Moreover, these molecule classes demonstrated synergy in combination with standard AML chemotherapy agents and activity in an orthotopic model of AML1-ETO-positive AML. This work suggests a role for DHFR inhibitors and corticosteroids in treating patients with AML1-ETO-positive disease.

Human natural resistance-associated macrophage protein: cDNA cloning, chromosomal mapping, genomic organization, and tissue-specific expression.

Natural resistance to infection with unrelated intracellular parasites such as Mycobacteria, Salmonella, and Leishmania is controlled in the mouse by a single gene on chromosome 1, designated Bcg, Ity, or Lsh. A candidate gene for Bcg, designated natural resistance-associated macrophage protein (Nramp), has been isolated and shown to encode a novel macrophage-specific membrane protein, which is altered in susceptible animals. We have cloned and characterized cDNA clones corresponding to the human NRAMP gene. Nucleotide and predicted amino acid sequence analyses indicate that the human NRAMP polypeptide encodes a 550-amino acid residue membrane protein with 10-12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionary conserved consensus transport motif. Identification of genomic clones corresponding to human NRAMP indicates that the gene maps to chromosome 2q35 within a group of syntenic loci conserved with proximal mouse 1. The gene is composed of at least 15 exons, with several exons encoding discrete predicted structural domains of the protein. These studies have also identified an alternatively spliced exon encoded by an Alu element present within intron 4. Although this novel exon was found expressed in vivo, it would introduce a termination codon in the downstream exon V, resulting in a severely truncated protein. Northern blot analyses indicate that NRAMP mRNA expression is tightly controlled in a tissue-specific fashion, with the highest sites of expression being peripheral blood leukocytes, lungs, and spleen. Additional RNA expression studies in cultured cells identified the macrophage as a site of expression of human NRAMP and indicated that increased expression was correlated with an advanced state of differentiation of this lineage.

Validating a simple scoring system to predict malignancy and invasiveness of intraductal papillary mucinous neoplasms of the pancreas.

BACKGROUND: The objective of the present study was to identify reliable preoperative factors predicting malignancy or invasiveness of intraductal papillary mucinous neoplasm (IPMN) of the pancreas and the effectiveness of a diagnostic scoring system based on these factors. METHODS: Between August 1994 and December 2007, 204 patients underwent pancreatic resection for IPMN at a single institute. Medical records were reviewed retrospectively, and a new diagnostic scoring system for predicting malignant IPMN preoperatively was designed. RESULTS: Univariate analysis revealed nine significant predictors of both malignant and invasive IPMN: age > or =60 years, history of pancreatitis, presence of mural nodule(s), diameter of main pancreatic duct (MPD) >6 mm, main duct or mixed type, total bilirubin >1.2 mg/dl, CA-19-9 >37 U/ml, tumor location in the pancreatic head, and tumor size >30 mm. Multivariate analysis showed that age, pancreatitis, mural nodule(s), and MPD diameter were independent predictors of invasive IPMN, and that all these parameters, plus elevated carbohydrate antigen-19-9 (CA-19-9), were independent predictors of malignant IPMN. A scoring system based on these five factors, each assigned 1 point, and with a cut-off of 3 points, could predict malignant IPMN with a sensitivity of 50.7% and a specificity of 90.1%. The 5-year survival rates of patients with benign and malignant IPMN were 95.0% and 64.0%, respectively. CONCLUSIONS: Our scoring system using five independent factors (age > or =60 years, history of pancreatitis, presence of mural nodule(s), elevated level of CA-19-9, and MPD diameter >6 mm) may be helpful for predicting malignancy and postoperative survival.

p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum.

We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.