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.

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.

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.

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.

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.

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.

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.

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.

Genomic prediction of relapse in recipients of allogeneic haematopoietic stem cell transplantation.

Allogeneic haematopoietic stem cell transplantation currently represents the primary potentially curative treatment for cancers of the blood and bone marrow. While relapse occurs in approximately 30% of patients, few risk-modifying genetic variants have been identified. The present study evaluates the predictive potential of patient genetics on relapse risk in a genome-wide manner. We studied 151 graft recipients with HLA-matched sibling donors by sequencing the whole-exome, active immunoregulatory regions, and the full MHC region. To assess the predictive capability and contributions of SNPs and INDELs, we employed machine learning and a feature selection approach in a cross-validation framework to discover the most informative variants while controlling against overfitting. Our results show that germline genetic polymorphisms in patients entail a significant contribution to relapse risk, as judged by the predictive performance of the model (AUC = 0.72 [95% CI: 0.63-0.81]). Furthermore, the top contributing variants were predictive in two independent replication cohorts (n = 258 and n = 125) from the same population. The results can help elucidate relapse mechanisms and suggest novel therapeutic targets. A computational genomic model could provide a step toward individualized prognostic risk assessment, particularly when accompanied by other data modalities.

IRF5 regulates unique subset of genes in dendritic cells during West Nile virus infection.

Pathogen recognition receptor (PRR) signaling is critical for triggering innate immune activation and the expression of immune response genes, including genes that impart restriction against virus replication. RIG-I-like receptors and TLRs are PRRs that signal immune activation and drive the expression of antiviral genes and the production of type I IFN leading to induction of IFN-stimulated genes, in part through the interferon regulatory factor (IRF) family of transcription factors. Previous studies with West Nile virus (WNV) showed that IRF3 and IRF7 regulate IFN expression in fibroblasts and neurons, whereas macrophages and dendritic cells (DCs) retained the ability to induce IFN-ß in the absence of IRF3 and IRF7 in a manner implicating IRF5 in PRR signaling actions. Here we assessed the contribution of IRF5 to immune gene induction in response to WNV infection in DCs. We examined IRF5-dependent gene expression and found that loss of IRF5 in mice resulted in modest and subtle changes in the expression of WNV-regulated genes. Anti-IRF5 chromatin immunoprecipitation with next-generation sequencing of genomic DNA coupled with mRNA analysis revealed unique IRF5 binding motifs within the mouse genome that are distinct from the canonical IRF binding motif and that link with IRF5-target gene expression. Using integrative bioinformatics analyses, we identified new IRF5 primary target genes in DCs in response to virus infection. This study provides novel insights into the distinct and unique innate immune and immune gene regulatory program directed by IRF5.

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.

Effects of asymmetry and hot-spot shape on ignition capsules.

Asymmetric implosion of inertial confinement fusion capsules is known, both experimentally and computationally, to reduce thermonuclear performance. This work shows that low-mode asymmetries degrade performance as a result of a decrease in the hydrodynamic disassembly time of the hot-spot core, which scales with the minimum dimension of the hot spot. The asymmetric shape of a hot spot results in decreased temperatures and areal densities and allows more alpha particles to escape, relative to an ideal spherical implosion, thus reducing alpha-energy deposition in the hot spot. Here, we extend previous ignition theory to include the hot-spot shape and quantify the effects of implosion asymmetry on both the ignition criterion and the capsule performance. The ignition criterion becomes more stringent with increasing deformation of the hot spot. The new theoretical results are validated by comparison with existing experimental data obtained at the National Ignition Facility. The shape effects on thermonuclear performance are relatively more noticeable for capsules having self-heating and high yields. The degradation of thermonuclear burn can be as high as 45% for shots with a yield lower than 2×10^{15} and less than 30% for shots with a higher yield.

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.

A Particle X-ray Temporal Diagnostic (PXTD) for studies of kinetic, multi-ion effects, and ion-electron equilibration rates in Inertial Confinement Fusion plasmas at OMEGA (invited).

A Particle X-ray Temporal Diagnostic (PXTD) has been implemented on OMEGA for simultaneous time-resolved measurements of several nuclear products as well as the x-ray continuum produced in High Energy Density Plasmas and Inertial Confinement Fusion implosions. The PXTD removes systematic timing uncertainties typically introduced by using multiple instruments, and it has been used to measure DD, DT, D3He, and T3He reaction histories and the emission history of the x-ray core continuum with relative timing uncertainties within ±10-20 ps. This enables, for the first time, accurate and simultaneous measurements of the x-ray emission histories, nuclear reaction histories, their time differences, and measurements of Ti(t) and Te(t) from which an assessment of multiple-ion-fluid effects, kinetic effects during the shock-burn phase, and ion-electron equilibration rates can be made.

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.

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.

Gfi1 and gfi1b repress rag transcription in plasmacytoid dendritic cells in vitro.

Growth factor independence genes (Gfi1 and Gfi1b) repress recombination activating genes (Rag) transcription in developing B lymphocytes. Because all blood lineages originate from hematopoietic stem cells (HSCs) and different lineage progenitors have been shown to share transcription factor networks prior to cell fate commitment, we hypothesized that GFI family proteins may also play a role in repressing Rag transcription or a global lymphoid transcriptional program in other blood lineages. We tested the level of Rag transcription in various blood cells when Gfi1 and Gfi1b were deleted, and observed an upregulation of Rag expression in plasmacytoid dendritic cells (pDCs). Using microarray analysis, we observed that Gfi1 and Gfi1b do not regulate a lymphoid or pDC-specific transcriptional program. This study establishes a role for Gfi1 and Gfi1b in Rag regulation in a non-B lineage cell type.

MK5 activates Rag transcription via Foxo1 in developing B cells.

Foxo1 is a critical, direct regulator of Rag (recombination activating gene) transcription during B cell development and is thus essential for the generation of a diverse repertoire of antigen receptors. Although Foxo1 regulation has been widely studied in many cell types, pathways regulating Foxo1 in B cells have not been fully elucidated. By screening a panel of Foxo1 mutants, we identified serine 215 on Foxo1 as a novel phosphorylation site that is essential for the activation of Rag transcription. Mutation of S215 strongly attenuated transactivation of Rag but did not affect most other Foxo1 target genes. We show that MK5, a MAPK-activated protein kinase, is a previously unidentified upstream regulator of Foxo1. MK5 was necessary and sufficient to activate Rag transcription in transformed and primary pro-B cells. Together, our experiments show that MK5 positively regulates Rag transcription via phosphorylation of Foxo1 in developing B cells.