Single-cell transcriptomics reveals variations in monocytes and Tregs between gout flare and remission.

Gout commonly manifests as a painful, self-limiting inflammatory arthritis. Nevertheless, the understanding of the inflammatory and immune responses underlying gout flares and remission remains ambiguous. Here, based on single-cell RNA-Seq and an independent validation cohort, we identified the potential mechanism of gout flare, which likely involves the upregulation of HLA-DQA1+ nonclassical monocytes and is related to antigen processing and presentation. Furthermore, Tregs also play an essential role in the suppressive capacity during gout remission. Cell communication analysis suggested the existence of altered crosstalk between monocytes and other T cell types, such as Tregs. Moreover, we observed the systemic upregulation of inflammatory and cytokine genes, primarily in classical monocytes, during gout flares. All monocyte subtypes showed increased arachidonic acid metabolic activity along with upregulation of prostaglandin-endoperoxide synthase 2 (PTGS2). We also detected a decrease in blood arachidonic acid and an increase in leukotriene B4 levels during gout flares. In summary, our study illustrates the distinctive immune cell responses and systemic inflammation patterns that characterize the transition from gout flares to remission, and it suggests that blood monocyte subtypes and Tregs are potential intervention targets for preventing recurrent gout attacks and progression.

Hybrid mRNA Nano Vaccine Potentiates Antigenic Peptide Presentation and Dendritic Cell Maturation for Effective Cancer Vaccine Therapy and Enhances Response to Immune Checkpoint Blockade.

Cancer vaccines combined with immune checkpoint blockades (ICB) represent great potential application, yet the insufficient tumor antigen presentation and immature dendritic cells hinder improved efficacy. Here, a hybrid nano vaccine composed by hyper branched poly(beta-amino ester), modified iron oxide nano adjuvant and messenger RNA (mRNA) encoded with model antigen ovalbumin (OVA) is presented. The nano vaccine outperforms three commercialized reagents loaded with the same mRNA, including Lipofectamine MessengerMax, jetPRIME, and in vivo-jetRNA in promoting dendritic cells' transfection, maturation, and peptide presentation. In an OVA-expressing murine model, intratumoral administration of the nano vaccine significantly induced macrophages and dendritic cells' presenting peptides and expressing co-stimulatory CD86. The nano vaccine also elicited strong antigen-specific splenocyte response and promoted CD8+ T cell infiltration. In combination with ICB, the nano vaccine aroused robust tumor suppression in murine models with large tumor burdens (initial volume >300 mm3 ). The hybrid mRNA vaccine represents a versatile and readily transformable platform and augments response to ICB.

MSU crystal deposition contributes to inflammation and immune responses in gout remission.

As a prominent feature of gout, monosodium urate (MSU) crystal deposition induces gout flares, but its impact on immune inflammation in gout remission remains unclear. Using single-cell RNA sequencing (scRNA-seq), we characterize the transcription profiling of peripheral blood mononuclear cells (PBMCs) among intercritical remission gout, advanced remission gout, and normal controls. We find systemic inflammation in gout remission with MSU crystal deposition at the intercritical and advanced stages, evidenced by activated inflammatory pathways, strengthened inflammatory cell-cell interactions, and elevated arachidonic acid metabolic activity. We also find increased HLA-DQA1high classic monocytes and PTGS2high monocytes in advanced gout and overactivated CD8+ T cell subtypes in intercritical and advanced gout. Additionally, the osteoclast differentiation pathway is significantly enriched in monocytes, T cells, and B cells from advanced gout. Overall, we demonstrate systemic inflammation and distinctive immune responses in gout remission with MSU crystal deposition, allowing further exploration of the underlying mechanism and clinical significance in conversion from intercritical to advanced stage.

Multiplex Digital Immunoassays with Reduced Pre-partition Reaction via Massively Parallel Reagent Delivery on a Bead-Based SlipChip.

The emergence of digital immunoassays has advanced the sensitivity of protein analysis to ultrahigh sensitivity at the attomolar level. However, the background signal generated by the premixing of immunocomplexes and fluorogenic substrates can limit the precise quantification, especially in multiplexed assays. Herein, a bead-based SlipChip (bb-SlipChip) microfluidic device capable of massively parallel two-step sample loading is presented. The background signal can be suppressed through a two-step loading mechanism. Specifically, encapsulate the beads into the microwells first and then, through a slipping process, deliver the fluorogenic substrate in parallel into 281,200 microwells of 68 fL to perform the digital immunoassay. The quantification capability is demonstrated with a duplex assay of IL-6 and IL-10, achieving a limit of detection of 5.2 and 15.3 fg/mL, which is approximately 2-3 times improved compared to a commercial Simoa system. The bb-SlipChip provides a robust and universal method for digital immunoassay and can be extended to higher multiplexed detection as well as other biomedical applications involving microbeads.

Emerging advances in nanobiomaterials-assisted chimeric antigen receptor (CAR)-macrophages for tumor immunotherapy.

Adoptive cell immunotherapy, especially chimeric antigen receptor (CAR)-T-cells therapy, has made great progress in the clinical treatment of hematological malignancies. However, restricted by the complex tumor microenvironment, the potential efficiency of T-cell infiltration and activated immune cells are limited, thus failure prevented the progression of the solid tumor. Alternatively, tumor-associated macrophages (TAMs), one sustentacular and heterogeneous cellular population within the tumor microenvironment, are regarded as potential therapeutic targets. Recently, CARs have shown tremendous promise in treating malignancies by equipping macrophages. This novel therapeutic strategy circumvents the tumor microenvironment's limitations and provides a safer therapeutic approach. Meanwhile, nanobiomaterials as gene delivery carriers not only substantially reduce the treatment cost of this novel therapeutic strategy, but also set the foundation for in vivo CAR-M therapy. Here, we highlight the major strategies prepared for CAR-M, emphasizing the challenges and opportunities of these approaches. First, the common therapeutic strategies for macrophages are summarized in clinical and preclinical trials. Namely, TAM-targeted therapeutic strategies: 1) Inhibit monocyte or macrophage recruitment into tumors, 2) deplete TAMs, and 3) reprogramme TAMs to antitumor M1 phenotype. Second, the current development and progress of CAR-M therapy are reviewed, including the researchers' attempts in CAR structure design, cell origin, and gene delivery vectors, especially nanobiomaterials as an alternative to viral vectors, as well as some challenges faced by current CAR-M therapy are also summarized and discussed. Finally, the field of genetically engineered macrophages integration with nanotechnology for the future in oncology has been prospected.

A micro-chamber free digital bio-detection for multiplexed and ultrasensitive immunoassay based on encoded magnetic microbeads and tyramide signal amplification strategy.

Digital bio-detection has become one of the most appealing methods in recent years due to its excellent performance with ultra-sensitivity in detection of low-abundance targets. Traditional digital bio-detection needs the utilization of micro-chambers for physical isolation of targets, while the recently developed beads-based micro-chamber free one is attracting extensive attention, although there exist the disadvantages of overlaps between positive ("1") and negative ("0") signals as well as the decreased detection sensitivity in multiplexed mode. Here we propose a feasible and robust micro-chamber free digital bio-detection for multiplexed and ultrasensitive immunoassay based on encoded magnetic microbeads (EMMs) and tyramide signal amplification (TSA) strategy. An EMMs-based multiplexed platform is constructed by using a fluorescent encoding method, then a puissant signal amplification of positive events in TSA procedure is achieved via systematical revelation of key factors influences. For proof of concept, a three-plexed tumor markers detection is performed to evaluate our established platform. The detection sensitivity is comparable to the corresponding single-plexed assays and is also approximately 30-15,000 times improvement compared to the conventional suspension chip. Therefore, this multiplexed micro-chamber free digital bio-detection paves a promising way to be an ultrasensitive and powerful tool for clinical diagnosis.

Joint Application of Multiple Inflammatory Cytokines in Diagnosis of Gout Flare.

Purpose: This study aimed to explore the accuracy for joint application of inflammatory cytokines in diagnosis of gout flare by comparison with peripheral blood cells. Methods: We collected the clinical data of 96 acute gout patients and 144 remission gout patients, and compared the levels of peripheral blood cells, inflammatory cytokines and blood biochemistry indexes between acute and remission gout. We respectively assessed the area under curves (AUCs) for single and multiple inflammatory cytokines including C-reactive protein (CRP), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and single and multiple peripheral blood cells including platelet (PLT), white blood cell (WBC), percentages of neutrophils (N%), lymphocytes (L%), eosinophils (E%), basophils (B%) in diagnosis of acute gout by receiver operating characteristic (ROC) curve analysis. Results: By contrast with remission gout, the levels of PLT, WBC, N%, CRP, IL-1ß, IL-6 and TNF-α increased, and the levels of L%, E% and B% decreased in acute gout. The AUCs of PLT, WBC, N%, L%, E% and B% in diagnosis of acute gout were respectively 0.591, 0.601, 0.581, 0.567, 0.608 and 0.635, while the AUC for joint application of these peripheral blood cells was 0.674. Moreover, the AUCs of CRP, IL-1ß, IL-6 and TNF-α in diagnosis of acute gout were respectively 0.814, 0.683, 0.622 and 0.746, while the AUC for joint application of these inflammatory cytokines was 0.883, reflecting significantly higher levels than peripheral blood cells. Conclusion: The joint application of multiple inflammatory cytokines can better distinguish acute gout from remission gout compared with peripheral blood cells.

Sequence terminus dependent PCR for site-specific mutation and modification detection.

The detection of changes in nucleic acid sequences at specific sites remains a critical challenge in epigenetics, diagnostics and therapeutics. To date, such assays often require extensive time, expertise and infrastructure for their implementation, limiting their application in clinical settings. Here we demonstrate a generalizable method, named Specific Terminal Mediated Polymerase Chain Reaction (STEM-PCR) for the detection of DNA modifications at specific sites, in a similar way as DNA sequencing techniques, but using simple and widely accessible PCR-based workflows. We apply the technique to both for site-specific methylation and co-methylation analysis, importantly using a bisulfite-free process - so providing an ease of sample processing coupled with a sensitivity 20-fold better than current gold-standard techniques. To demonstrate the clinical applicability through the detection of single base mutations with high sensitivity and no-cross reaction with the wild-type background, we show the bisulfite-free detection of SEPTIN9 and SFRP2 gene methylation in patients (as key biomarkers in the prognosis and diagnosis of tumours).

Multiplexed and accurate quantification strategy for miRNA based on specific terminal-mediated PCR with equivalent amplification.

MicroRNAs (miRNAs) are recognized as potential biomarkers for the early diagnosis and prognosis of different diseases. Multiplexed and accurate miRNA quantification methods with equivalent detection efficiency are particularly crucial due to their complex biological functions and lack of a unified internal reference gene. Here, a unique multiplexed miRNA detection method, named Specific Terminal-Mediated miRNA PCR (STEM-Mi-PCR), was developed. It mainly includes a linear reverse transcription step using tailored-designed target specific capture primers, followed by an exponential amplification process using two universal primers to execute the multiplex assay. For proof of concept, four miRNAs were used as models to develop a multiplexed detection assay within one tube simultaneously and then evaluate the performance of the established STEM-Mi-PCR. The sensitivity of the 4-plexed assay was approximately 100 aM with an equivalent amplification efficiency (95.67 ± 8.58%), and had no cross-reactivity each other with high specificity. Quantification of different miRNAs in twenty patients' tissues shown variation from approximately pM to fM concentration level, demonstrating the possibility of practical application of the established method. Moreover, this method was extraordinarily capable of single nucleotide mutation discrimination in different let-7 family members with no more than 0.7% nonspecific detection signal. Hence, the STEM-Mi-PCR we proposed here paves an easy and promising way for miRNA profiling in future clinical applications.

Cell membrane-camouflaged liposomes and neopeptide-loaded liposomes with TLR agonist R848 provides a prime and boost strategy for efficient personalized cancer vaccine therapy.

Recent advances in bioinformatics and nanotechnology offer great opportunities for personalized cancer vaccine development. However, the timely identification of neoantigens and unsatisfactory efficacy of therapeutic cancer vaccines remain two obstacles for clinical transformation. We propose a "prime and boost" strategy to facilitate neoantigen-based immunotherapy. To prime the immune system, we first constructed personalized liposomes with cancer cell membranes and adjuvant R848 to provide immunostimulatory efficacy and time for identifying tumor antigens. Liposomes loaded with personalized neopeptides and adjuvants were used to boost the immune response. In vitro experiments verified potent immune responses, including macrophage polarization, dendritic cell maturation, and T lymphocyte activation. In vivo B16F10 and TC-1 cancer model were used to investigate efficient tumor growth suppression. Liposomal vaccines with neopeptides could stimulate human dendritic cells and T lymphocytes in vitro. These results demonstrate that the "prime and boost" strategy provides simple, quick, and efficient personalized vaccines for cancer therapy.

Mannose and Hyaluronic Acid Dual-Modified Iron Oxide Enhances Neoantigen-Based Peptide Vaccine Therapy by Polarizing Tumor-Associated Macrophages.

Neoantigen-based cancer vaccine therapy is a breakthrough in the field of immunotherapy. However, it is difficult for vaccines against neoantigens to overcome the immunosuppressive microenvironment, where tumor-associated macrophages (TAMs) play a significant role. Herein, we report an iron oxide nanoparticle modified with hyaluronic acid and mannose to reshape the tumor microenvironment by targeting and repolarizing TAMs from protumor M2 to antitumor M1 phenotype. Mannose decoration could confer the nanoparticle-enhanced TAM targeting ability, while hyaluronic acid and iron oxide could repolarize M2-like macrophages both in vitro and in vivo. Combined with antigenic peptides, this nanovaccine could significantly increase the infiltration of CD8+ T cells into tumor tissue and strongly activate dendritic cells in sentinel lymph nodes. Finally, we used the dual-modified nanoparticles to first convert the tumor microenvironment and then the nanovaccine administration in a TC1 tumor model to further enhance efficacy. This strategy inhibited tumor growth and achieved a 40% cure rate in mice (two of five). In summary, this study provides a potent and rationally designed nanoadjuvant to enhance antitumor efficiency and facilitate delivery of neoantigen vaccines by repolarizing TAMs and harmonizing immune cells.

Ultrasensitive monitoring of SARS-CoV-2-specific antibody responses based on a digital approach reveals one week of IgG seroconversion.

COVID-19 is still unfolding, while many people have been vaccinated. In comparison to nucleic acid testing (NAT), antibody-based immunoassays are faster and more convenient. However, its application has been hampered by its lower sensitivity and the existing fact that by traditional immunoassays, the measurable seroconversion time of pathogen-specific antibodies, such as IgM or IgG, lags far behind that of nucleic acids. Herein, by combining the single molecule array platform (Simoa), RBD, and a previously identified SARS-CoV-2 S2 protein derivatized 12-aa peptide (S2-78), we developed and optimized an ultrasensitive assay (UIM-COVID-19 assay). Sera collected from three sources were tested, i.e., convalescents, inactivated virus vaccine-immunized donors and wild-type authentic SARS-CoV-2-infected rhesus monkeys. The sensitivities of UIM-COVID-19 assays are 100-10,000 times higher than those of conventional flow cytometry, which is a relatively sensitive detection method at present. For the established UIM-COVID-19 assay using RBD as a probe, the IgG and IgM seroconversion times after vaccination were 7.5 and 8.6 days vs. 21.4 and 24 days for the flow cytometry assay, respectively. In addition, using S2-78 as a probe, the UIM-COVID-19 assay could differentiate COVID-19 patients (convalescents) from healthy people and patients with other diseases, with AUCs ranging from 0.85-0.95. In summary, the UIM-COVID-19 we developed here is a promising ultrasensitive biodetection strategy that has the potential to be applied for both immunological studies and diagnostics.

Bead-Based Multiplexed Droplet Digital Polymerase Chain Reaction in a Single Tube Using Universal Sequences: An Ultrasensitive, Cross-Reaction-Free, and High-Throughput Strategy.

The multiplexed digital polymerase chain reaction (PCR) is widely used in molecular diagnosis owing to its high sensitivity and throughput for multiple target detection compared with the single-plexed digital PCR; however, current multiplexed digital PCR technologies lack efficient coding strategies that do not compromise the sensitivity and signal-to-noise (S/N) ratio. Hence, we propose a fluorescent-encoded bead-based multiplexed droplet digital PCR method for ultra-high coding capacity, along with the creative design of universal sequences (primer and fluorescent TaqMan probe) for ultra-sensitivity and high S/N ratios. First, pre-amplification is used to introduce universal primers and universal fluorescent TaqMan probes to reduce primer interference and background noise, as well as to enrich regions of interest in targeted analytes. Second, fluorescent-encoded beads (FEBs), coupled with the corresponding target sequence-specific capture probes through streptavidin-biotin conjugation, are used to partition amplicons via hybridization according to the Poisson distribution. Finally, FEBs mixed with digital PCR mixes are isolated into droplets generated via Sapphire chips (Naica Crystal Digital PCR system) to complete the digital PCR and result analysis. For proof of concept, we demonstrate that this method achieves high S/N ratios in a 5-plexed assay for influenza viruses and SARS-CoV-2 at concentrations below 10 copies and even close to a single molecule per reaction without cross-reaction, further verifying the possibility of clinical actual sample detection with 100% accuracy, which paves the way for the realization of digital PCR with ultrahigh coding capacity and ultra-sensitivity.

Sensitive GlaI digestion and terminal transferase PCR for DNA methylation detection.

Highly sensitive and specific detection of DNA methylation is critical for early diagnosis and therapy of cancer. Herein, we propose a novel bisulfite-free PCR assay based on a GlaI methylation specific digestion and terminal transferase (TdT) extension for the detection of methylated DNA with high sensitivity and specificity, denoted as GlaI-TdT methylation PCR. For GlaI-TdT methylation PCR assay, the methylated CpG site is recognized and cut by GlaI selectively firstly, leading to the generation of product with specific free 3' end. The free 3' end can be further extended with TdT and served as template for the followed quantitative PCR. The specificity of GlaI-TdT methylation PCR depends on the specific methylation discrimination of GlaI and the existence of poly-T sequence as the extension of TdT. The sensitivity of GlaI-TdT PCR for methylated DNA can achieve 10 copies/reaction with 10,000 copies unmethylated background. The detection performance of GlaI-TdT methylation PCR was also evaluated using colorectal cancer tissue samples, with the results shown great accordance with standard bisulfite-PCR sequencing. Based on its high sensitivity, high specificity, simple and convenient, GlaI-TdT methylation PCR has the great potential to become a promising and robust bisulfite-free procedure for the detection of DNA methylations.

Breaking through the Poisson Distribution: A compact high-efficiency droplet microfluidic system for single-bead encapsulation and digital immunoassay detection.

Droplet encapsulation of a single cell or bead is widely used in digital detection, single-cell sequencing, and drug screening. However, the encapsulation of particles is totally random restricted by the Poisson distribution. The theoretical possibility of single-particle encapsulation is usually only approximately 10%. In ultra-high multiplexed digital detection or other applications that needing to measure large numbers of particles, the number of the partitions required to be counted is extremely high, further result in great increase of statistical number of invalid droplets and the redundancy of detection data. Here, a bead ordered arrangement droplet (BOAD) system is proposed to break through the Poisson distribution. BOAD system tactfully combines sheath flow, Dean vortex, and compression flow channel to achieve orderly arrangement of particles for the first time, and could achieve the fastest orderly arrangement of particles in the shortest structure. The efficiency of single-bead encapsulation is improved to as high as 86%. Further application to encapsulate encoding beads and IL-10-targeted magnetic beads demonstrates the potential for bead-based ultra-high multiplexed digital detection. Thus, use of the BOAD system is very promising for many applications needing high single-particle encapsulation ratio in limited partitions, such as multiplexed digital bio-detection, single-cell analysis, drug screening, and single exosome detection.

Programmable design of isothermal nucleic acid diagnostic assays through abstraction-based models.

Accelerating the design of nucleic acid amplification methods remains a critical challenge in the development of molecular tools to identify biomarkers to diagnose both infectious and non-communicable diseases. Many of the principles that underpin these mechanisms are often complex and can require iterative optimisation. Here we focus on creating a generalisable isothermal nucleic acid amplification methodology, describing the systematic implementation of abstraction-based models for the algorithmic design and application of assays. We demonstrate the simplicity, ease and flexibility of our approach using a software tool that provides amplification schemes de novo, based upon a user-input target sequence. The abstraction of reaction network predicts multiple reaction pathways across different strategies, facilitating assay optimisation for specific applications, including the ready design of multiplexed tests for short nucleic acid sequence miRNAs or for difficult pathogenic targets, such as highly mutating viruses.

Evolution of "On-Barcode" Luminescence Oxygen Channeling Immunoassay by Exploring the Barcode Structure and the Assay System.

The multiplexed luminescence oxygen channeling immunoassay (multi-LOCI) platform we developed recently that combines conventional LOCI and suspension array technology is capable of realizing facile "mix-and-measure" multiplexed assays without tedious washing steps. However, previous work lacks comprehensive studies of the structure-performance relationship of the host-guest-structured barcode, which may obstruct the evolution and further translation of this exciting new technology to practical applications. Accordingly, this work revealed that polyelectrolyte interlayers played a crucial role in tuning the packing density of guest acceptor beads (ABs). More interestingly, we noticed that "sparse" barcodes (barcodes with low ABs packing density) exhibited comparable assay performance with "compact" ones (barcodes with high ABs packing density). The high robustness of barcodes allows for multi-LOCI to be a more universal and flexible assay platform. Furthermore, through optimization of the assay system including the laser power, as well as the concentrations of donor beads and biotinylated detection antibodies, the multi-LOCI platform showed a significant improvement in sensitivity compared with our previous work, with the limit of detection decreasing to as low as ca. 1 pg/mL. Impressively, multi-LOCI that enabled simultaneous detection of multiple analytes exhibited comparable sensitivity with the classical single-plexed LOCI, due to the ingenious structural design of the multi-LOCI barcode and the unique "on-barcode" assay format.

Emerging Nanoparticle Strategies for Modulating Tumor-Associated Macrophage Polarization.

Immunotherapy has made great progress in recent years, yet the efficacy of solid tumors remains far less than expected. One of the main hurdles is to overcome the immune-suppressive tumor microenvironment (TME). Among all cells in TME, tumor-associated macrophages (TAMs) play pivotal roles because of their abundance, multifaceted interactions to adaptive and host immune systems, as well as their context-dependent plasticity. Underlying the highly plastic characteristic, lots of research interests are focused on repolarizing TAMs from M2-like pro-tumor phenotype towards M1-like antitumoral ones. Nanotechnology offers great opportunities for targeting and modulating TAM polarization to mount the therapeutic efficacy in cancer immunotherapy. Here, this mini-review highlights those emerging nano-approaches for TAM repolarization in the last three years.