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Progress in the development of effective chemotherapy is producing a growing population of patients with acute and chronic painful chemotherapy-induced peripheral neuropathy (CIPN), a serious treatment-limiting side effect for which there is currently no US Food and Drug Administration-approved treatment. CIPNs induced by diverse classes of chemotherapy drugs have remarkably similar clinical presentations, leading to the suggestion they share underlying mechanisms. Sensory neurons share with immune cells the ability to detect damage associated molecular patterns (DAMPs), molecules produced by diverse cell types in response to cellular stress and injury, including by chemotherapy drugs. DAMPs, in turn, are ligands for pattern recognition receptors (PRRs), several of which are found on sensory neurons, as well as satellite cells, and cells of the immune system. In the present experiments, we evaluated the role of two PRRs, TLR4 and RAGE, present in dorsal root ganglion (DRG), in CIPN. Antisense (AS)-oligodeoxynucleotides (ODN) against TLR4 and RAGE mRNA were administered intrathecally before ('prevention protocol') or 3 days after ('reversal protocol') the last administration of each of three chemotherapy drugs that treat cancer by different mechanisms (oxaliplatin, paclitaxel and bortezomib). TLR4 and RAGE AS-ODN prevented the development of CIPN induced by all three chemotherapy drugs. In the reversal protocol, however, while TLR4 AS-ODN completely reversed oxaliplatin- and paclitaxel-induced CIPN, in rats with bortezomib-induced CIPN it only produced a temporary attenuation. RAGE AS-ODN, in contrast, reversed CIPN induced by all three chemotherapy drugs. When a TLR4 antagonist was administered intradermally to the peripheral nociceptor terminal, it did not affect CIPN induced by any of the chemotherapy drugs. However, when administered intrathecally, to the central terminal, it attenuated hyperalgesia induced by all three chemotherapy drugs, compatible with a role of TLR4 in neurotransmission at the central terminal but not sensory transduction at the peripheral terminal. Finally, since it has been established that cultured DRG neurons can be used to study direct effects of chemotherapy on nociceptors, we also evaluated the role of TLR4 in CIPN at the cellular level, using patch-clamp electrophysiology in DRG neurons cultured from control and chemotherapy-treated rats. We found that increased excitability of small-diameter DRG neurons induced by in vivo and in vitro exposure to oxaliplatin is TLR4-dependent. Our findings suggest that in addition to the established contribution of PRR-dependent neuroimmune mechanisms, PRRs in DRG cells also have an important role in CIPN. [ABSTRACT FROM AUTHOR]

Cyclic dinucleotides (CDN) and Toll-like receptor (TLR) ligands mobilize antitumor responses by natural killer (NK) cells and T cells, potentially serving as complementary therapies to immune checkpoint therapy. In the clinic thus far, however, CDN therapy targeting stimulator of interferon genes (STING) protein has yielded mixed results, perhaps because it initiates responses potently but does not provide signals to sustain activation and proliferation of activated cytotoxic lymphocytes. To improve efficacy, we combined CDN with a half life-extended interleukin-2 (IL-2) superkine, H9-MSA (mouse serum albumin). CDN/H9-MSA therapy induced dramatic long-term remissions of the most difficult to treat major histocompatibility complex class I (MHC I)–deficient and MHC I+ tumor transplant models. H9-MSA combined with CpG oligonucleotide also induced potent responses. Mechanistically, tumor elimination required CD8 T cells and not NK cells in the case of MHC I+ tumors and NK cells but not CD8 T cells in the case of MHC-deficient tumors. Furthermore, combination therapy resulted in more prolonged and more intense NK cell activation, cytotoxicity, and expression of cytotoxic effector molecules in comparison with monotherapy. Remarkably, in a primary autochthonous sarcoma model that is refractory to PD-1 checkpoint therapy, the combination of CDN/H9-MSA with checkpoint therapy yielded long-term remissions in the majority of the animals, mediated by T cells and NK cells. This combination therapy has the potential to activate responses in tumors resistant to current therapies and prevent MHC I loss accompanying acquired resistance of tumors to checkpoint therapy.

Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants.

High intensity focused ultrasound (HIFU) rapidly and non-invasively destroys tumor tissue. Here, we sought to assess the immunomodulatory effects of MR-guided HIFU and its combination with the innate immune agonist CpG and checkpoint inhibitor anti-PD-1. Mice with multi-focal breast cancer underwent ablation with a parameter set designed to achieve mechanical disruption with minimal thermal dose or a protocol in which tumor temperature reached 65 °C. Mice received either HIFU alone or were primed with the toll-like receptor 9 agonist CpG and the checkpoint modulator anti-PD-1. Both mechanical HIFU and thermal ablation induced a potent inflammatory response with increased expression of Nlrp3, Jun, Mefv, Il6 and Il1β and alterations in macrophage polarization compared to control. Furthermore, HIFU upregulated multiple innate immune receptors and immune pathways, including Nod1, Nlrp3, Aim2, Ctsb, Tlr1/2/4/7/8/9, Oas2, and RhoA. The inflammatory response was largely sterile and consistent with wound-healing. Priming with CpG attenuated Il6 and Nlrp3 expression, further upregulated expression of Nod2, Oas2, RhoA, Pycard, Tlr1/2 and Il12, and enhanced T-cell number and activation while polarizing macrophages to an anti-tumor phenotype. The tumor-specific antigen, cytokines and cell debris liberated by HIFU enhance response to innate immune agonists.

The molecular mechanisms that drive hematopoietic stem cell functional decline under conditions of telomere shortening are not completely understood. In light of recent advances in single-cell technologies, we sought to redefine the transcriptional and epigenetic landscape of mouse and human hematopoietic stem cells under telomere attrition, as induced by pathogenic germline variants in telomerase complex genes. Here, we show that telomere attrition maintains hematopoietic stem cells under persistent metabolic activation and differentiation towards the megakaryocytic lineage through the cell-intrinsic upregulation of the innate immune signaling response, which directly compromises hematopoietic stem cells' self-renewal capabilities and eventually leads to their exhaustion. Mechanistically, we demonstrate that targeting members of the Ifi20x/IFI16 family of cytosolic DNA sensors using the oligodeoxynucleotide A151, which comprises four repeats of the TTAGGG motif of the telomeric DNA, overcomes interferon signaling activation in telomere-dysfunctional hematopoietic stem cells and these cells' skewed differentiation towards the megakaryocytic lineage. This study challenges the historical hypothesis that telomere attrition limits the proliferative potential of hematopoietic stem cells by inducing apoptosis, autophagy, or senescence, and suggests that targeting IFI16 signaling axis might prevent hematopoietic stem cell functional decline in conditions affecting telomere maintenance.

Background: Spinal N-methyl-D-aspartate (NMDA) receptor activation is attributed to remifentanil-induced hyperalgesia (RIH). However, the specific mechanism and subsequent treatment is still unknown. Previous studies have shown that the dynamin-related protein 1 (DRP1)-mitochondria-reactive oxygen species (ROS) pathway plays an important role in neuropathic pain. This study examined whether antisense oligodeoxynucleotides against DRP1 (AS-DRP1) could reverse RIH. Methods: The authors first measured changes in paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) at 24 hours before remifentanil infusion and 4, 8, 24, and 48 hours after infusion. The expression levels of DRP1 and NR2B were measured after behavioral testing using Western blotting. In addition, DRP1 expression was knocked down by intrathecal administration of AS-DRP1 to investigate the effects of DRP1 on RIH. The behavioral testing, the expression levels of spinal DRP1 and NR2B, and dorsal mitochondrial superoxide were measured. Changes in mitochondrial morphology were assessed using electron microscopy. Results: After remifentanil exposure, upregulation of spinal DRP1 and NR2B was observed along with a reduction in PWMT and PWTL. In addition, AS-DRP1 improved RIH-induced PWTL and PWMT (P < 0.001 and P < 0.001) and reduced remifentanil-mediated enhancement of spinal DRP1 and NR2B expression (P = 0.020 and P = 0.022). More importantly, AS-DRP1 reversed RIH-induced mitochondrial fission (P = 0.020) and mitochondrial superoxide upregulation (P = 0.031). Conclusions: These results indicate that AS-DRP1 could modulate NMDA receptor expression to prevent RIH through the DRP1-mitochondria-ROS pathway. [ABSTRACT FROM AUTHOR]

A new vaccine formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) and amphipols was assessed in an intranasal (i.n.) challenge mouse model. nMOMP was trapped either in amphipol A8-35 (nMOMP/A8-35) or in A8-35 conjugated with Resiquimod (nMOMP/Resiq-A8-35), a TLR7/8 agonist added as adjuvant. The effects of free Resiquimod and/or additional adjuvants, Montanide ISA 720 (TLR independent) and CpG-1826 (TLR9 agonist), were also evaluated. Immunization with nMOMP/A8-35 alone administered i.n. was used as negative adjuvant-control group, whereas immunizations with C. muridarum elementary bodies (EBs) and MEM buffer, administered i.n., were used as positive and negative controls, respectively. Vaccinated mice were challenged i.n. with C. muridarum and changes in body weight, lungs weight and recovery of Chlamydia from the lungs were evaluated. All the experimental groups showed protection when compared with the negative control group. Resiquimod alone produced weak humoral and cellular immune responses, but both Montanide and CpG-1826 showed significant increases in both responses. The addition of CpG-1826 alone switched immune responses to be Th1-biased. The most robust protection was elicited in mice immunized with the three adjuvants and conjugated Resiquimod. Increased protection induced by the Resiquimod covalently linked to A8-35, in the presence of Montanide and CpG-1826 was established based on a set of parameters: (1) the ability of the antibodies to neutralize C. muridarum; (2) the increased proliferation of T-cells in vitro accompanied by higher production of IFN-γ, IL-6 and IL-17; (3) the decreased body weight loss over the 10 days after challenge; and (4) the number of IFUs recovered from the lungs at day 10 post challenge. In conclusion, a vaccine formulated with the C. muridarum nMOMP bound to amphipols conjugated with Resiquimod enhances protective immune responses that can be further improved by the addition of Montanide and CpG-1826.

Mass spectrometry and tandem MS (MS/MS) have been widely employed for the identification and quantification of damaged nucleosides in DNA, including those induced by alkylating agents. Upon collisional activation, protonated ions of alkylated nucleosides frequently undergo facile neutral loss of a 2-deoxyribose in MS/MS, and further cleavage of the resulting protonated nucleobases in MS3 can sometimes be employed for differentiating regioisomeric alkylated DNA lesions. Herein, we investigated systematically the collision-induced dissociation (CID) of the protonated ions of O4-alkylthymidine (O4-alkyldT), O2-alkyldT, O6-alkyl-2-deoxyguanosine (O6-alkyldG), and N2-alkyldG through MS3 analysis. The MS3 of O2- and O4-MedT exhibit different fragmentation patterns from each other and from other O2- and O4-alkyldT adducts carrying larger alkyl groups. Meanwhile, elimination of alkene via a six-membered ring transition state is the dominant fragmentation pathway for O2-alkyldT, O4-alkyldT, and O6-alkyldG adducts carrying larger alkyl groups, whereas O6-MedG mainly undergoes elimination of ammonia. The breakdown of N2-alkyldG is substantially influenced by the structure of the alkyl group, where the relative ease in eliminating ammonia and alkene is modulated by the chain length and branching of the alkyl groups. We also rationalize our observations with density functional theory (DFT) calculations.

Brain lipoprotein receptors have been shown to regulate the metabolism of ApoE and β-amyloid (Aβ) and are potential therapeutic targets for Alzheimer's disease (AD). Previously, we identified E3 ubiquitin ligase IDOL as a negative regulator of brain lipoprotein receptors. Genetic ablation of Idol increases low-density lipoprotein receptor protein levels, which facilitates Aβ uptake and clearance by microglia. In this study, we utilized an antisense oligonucleotide (ASO) to reduce IDOL expression therapeutically in the brains of APP/PS1 male mice. ASO treatment led to decreased Aβ pathology and improved spatial learning and memory. Single-cell transcriptomic analysis of hippocampus revealed that IDOL inhibition upregulated lysosomal/phagocytic genes in microglia. Furthermore, clustering of microglia revealed that IDOL-ASO treatment shifted the composition of the microglia population by increasing the prevalence of disease-associated microglia. Our results suggest that reducing IDOL expression in the adult brain promotes the phagocytic clearance of Aβ and ameliorates Aβ-dependent pathology. Pharmacological inhibition of IDOL activity in the brain may represent a therapeutic strategy for the treatment of AD.

Raman nanoparticle probes are a potent class of optical labels for the interrogation of pathological and physiological processes in cells, bioassays, and tissues. Herein, we review the recent advancements in fluorescent and Raman imaging using oligodeoxyribonucleotide (ODN)-based nanoparticles and nanostructures, which show promise as effective tools for live-cell analysis. These nanodevices can be used to investigate a vast number of biological processes occurring at various levels, starting from those involving organelles, cells, tissues, and whole living organisms. ODN-based fluorescent and Raman probes have contributed to the achievement of significant advancements in the comprehension of the role played by specific analytes in pathological processes and have inaugurated new possibilities for diagnosing health conditions. The technological implications that have emerged from the studies herein described could open new avenues for innovative diagnostics aimed at identifying socially relevant diseases like cancer through the utilization of intracellular markers and/or guide surgical procedures based on fluorescent or Raman imaging. Particularly complex probe structures have been developed within the past five years, creating a versatile toolbox for live-cell analysis, with each tool possessing its own strengths and limitations for specific studies. Analyzing the literature reports in the field, we predict that the development of ODN-based fluorescent and Raman probes will continue in the near future, disclosing novel ideas on their application in therapeutic and diagnostic strategies. [ABSTRACT FROM AUTHOR]

Recent advances in synthetic biology have led to the development of nucleic acid polymers with backbone structures distinct from those found in nature, termed xeno-nucleic acids (XNAs). Several unique properties of XNAs make them attractive as nucleic acid therapeutics, most notably their high resistance to serum nucleases and ability to form Watson-Crick base pairing with DNA and RNA. The ability of XNAs to induce immune responses has not been investigated. Threose nucleic acid (TNA), a type of XNA, is recalcitrant to nuclease digestion and capable of undergoing Darwinian evolution to produce high affinity aptamers; thus, TNA is an attractive candidate for diverse applications, including nucleic acid therapeutics. In this study, we evaluated a TNA oligonucleotide derived from a cytosine-phosphate-guanine oligonucleotide sequence known to activate toll-like receptor 9-dependent immune signaling in B cell lines. We observed a slight induction of relevant mRNA signals, robust B cell line activation, and negligible effects on cellular proliferation.

DisclosuresFunding for this summary was contributed by the Laura and John Arnold Foundation, Blue Shield of California, and California Health Care Foundation to the Institute for Clinical and Economic Review (ICER), an independent organization that evaluates the evidence on the value of health care interventions. ICER's annual policy summit is supported by dues from Aetna, AHIP, Anthem, Blue Shield of California, CVS Caremark, Express Scripts, Harvard Pilgrim Health Care, Cambia Health Solutions, United Healthcare, Kaiser Permanente, Premera Blue Cross, AstraZeneca, Genentech, GlaxoSmithKline, Johnson & Johnson, Merck, National Pharmaceutical Council, Prime Therapeutics, Sanofi, Spark Therapeutics, Health Care Service Corporation, Editas, Alnylam, Regeneron, Mallinkrodt, Biogen, HealthPartners, and Novartis. Mickle, Dreitlein, and Pearson are ICER employees. Lasser, Cipriano, and Hoch have nothing to disclose.

Hereditary transthyretin amyloidosis is a fatal autosomal dominant disorder characterized by deposition of transthyretin amyloid into the peripheral nervous system, heart, kidney, and gastrointestinal tract. Previous treatments using liver transplantation and small molecule stabilizers were not effective in stopping disease progression. Inotersen, a 2'-O-methyoxyethyl-modified antisense oligonucleotide, which acts by reducing the production of transthyretin, was recently demonstrated to improve disease course and quality of life in early hereditary transthyretin amyloidosis polyneuropathy in a 15-month Phase III study.

Antisense oligonucleotide (AON)-mediated exon skipping is an emerging therapeutic for individuals with Duchenne muscular dystrophy (DMD). Skipping of exons adjacent to common exon deletions in DMD using AONs can produce in-frame transcripts and functional protein. Targeted skipping of DMD exons 8, 44, 45, 50, 51, 52, 53, and 55 is predicted to benefit 47% of affected individuals. We observed a correlation between mutation subgroups and age at loss of ambulation in the Duchenne Registry, a large database of phenotypic and genetic data for DMD (N = 765). Males amenable to exon 44 (N = 74) and exon 8 skipping (N = 18) showed prolonged ambulation compared to other exon skip groups and nonsense mutations (P = 0.035 and P

Cyclic STAT3 decoy (CS3D) is a second-generation, double-stranded oligodeoxynucleotide (ODN) that mimics a genomic response element for signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor. CS3D competitively inhibits STAT3 binding to target gene promoters, resulting in decreased expression of proteins that promote cellular proliferation and survival. Previous studies have demonstrated antitumor activity of CS3D in preclinical models of solid tumors. However, prior to entering human clinical trials, the efficiency of generating the CS3D molecule and its stability in biological fluids should be determined. CS3D is synthesized as a single-stranded ODN and must have its free ends ligated to generate the final cyclic form. In this study, we report a ligation efficiency of nearly 95 percent. The ligated CS3D demonstrated a half-life of 7.9 h in human serum, indicating adequate stability for intravenous delivery. These results provide requisite biochemical characterization of CS3D that will inform upcoming clinical trials.

Nanoparticles have attracted considerable interest as cancer vaccine delivery vehicles for inducing sufficient CD8+ T cell-mediated immune responses to overcome the low immunogenicity of the tumor microenvironment. Our studies described here are the first to examine the effects of clinically-tested human cancer-testis (CT) peptide epitopes within a synthetic nanoparticle. Specifically, we focused on two significant clinical CT targets, the HLA-A2 restricted epitopes of NY-ESO-1 and MAGE-A3, using a viral-mimetic packaging strategy. Our data shows that simultaneous delivery of a NY-ESO-1 epitope (SLLMWITQV) and CpG using the E2 subunit assembly of pyruvate dehydrogenase (E2 nanoparticle), resulted in a 25-fold increase in specific IFN-γ secretion in HLA-A2 transgenic mice. This translated to a 15-fold increase in lytic activity toward target cancer cells expressing the antigen. Immunization with a MAGE-A3 epitope (FLWGPRALV) delivered with CpG in E2 nanoparticles yielded an increase in specific IFN-γ secretion and cell lysis by 6-fold and 9-fold, respectively. Furthermore, combined delivery of NY-ESO-1 and MAGE-A3 antigens in E2 nanoparticles yielded an additive effect that increased lytic activity towards cells bearing NY-ESO-1+ and MAGE-A3+. Our investigations demonstrate that formulation of CT antigens within a nanoparticle can significantly enhance antigen-specific cell-mediated responses, and the combination of the two antigens in a vaccine can preserve the increased individual responses that are observed for each antigen alone.

Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments. Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components. Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80, Cd11b and Tnf and the distant tumors to enhanced Cd11c, Cd3 and Ifng. When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to ~8% of CD45+ cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (~16% of CD45+ cells) compared to the ablation-only (~0.1% of CD45+ cells) and immunotherapy-only cohorts (~10% of CD45+ cells). Macrophages and DCs process and present this antigen to CD8+ T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and Icos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169+ macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation. Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response.

Molecular spherical nucleic acids (m-SNAs) are a second generation of spherical nucleic acids (SNAs), which are of significance in potential application of targeted delivery of nucleic acids or gene regulation due to their defined molecular structures. Nevertheless, m-SNAs typically involve a single DNA sequence which greatly limits its functions as either targeting purpose or gene regulation. In response, we proposed here a third generation, supramolecular spherical nucleic acids (Supra-SNAs) with two different sequences to achieve both above-mentioned functions. Specifically, we constructed a series of supramolecular self-assembly structures by coupling a cell membrane receptor (i.e., nucleolin)-recognizing aptamer (AS1411)-modified adamantine as targeting probe and human epithelial growth factor receptor 2 (HER2) antisense-functionalized β-cyclodextrin to specifically inhibit the overexpression of HER2 proteins for gene regulations. In comparison to the m-SNA precursors, such Supra-SNA structures exhibited enhanced levels of resistance to nuclease degradation, cellular uptake, gene regulation capabilities and tumor retention capacity. We demonstrated that Supra-SNAs exhibited optimal cell suppression rates and cell apoptosis via a phosphatidylinositol 3-kinase/protein kinase B signaling pathway. The well-defined molecular structures provide an attractive platform for investigating interrelationship between structure and property at the molecular level., (© 2024 Wiley-VCH GmbH.)

Messenger RNA (mRNA) vaccine has fueled a great hope for cancer immunotherapy. However, low immunogenicity, caused by inefficient mRNA expression and weak immune stimulation, hampers the efficacy of mRNA vaccines. Here, we present an mRNA compartmentalization-based cancer vaccine, comprising a multimodule DNA nanostructure (MMDNS)-assembled compartment for efficient mRNA translation via in situ localizing mRNA concentration and relevant reaction molecules. The MMDNS is constructed via programmable DNA hybridization chain reaction (HCR)-based strategy, with integrating antigen-coded mRNA, CpG oligodeoxynucleotides (ODNs), acidic-responsive DNA sequence, and dendritic cells targeting aptamer. MMDNS undergoes in situ assembly in acidic lysosomes to form a micro-sized aggregate, inducing an enhanced CpG ODN adjuvant efficacy. Subsequently, the aggregates escape into cytoplasm, providing a moderate compartment which supports the efficient translation of spatially proximal mRNA transcripts via localizing relevant reaction molecules. The mRNA compartmentalization-based vaccine boosts a strong immune response and effectively inhibits tumor growth and metastasis, offering a robust strategy for cancer immunotherapy.

Gene synthesis, the process of assembling gene-length fragments from shorter groups of oligonucleotides (oligos), is becoming an increasingly important tool in molecular and synthetic biology. The length, quality and cost of gene synthesis are limited by errors produced during oligo synthesis and subsequent assembly. Enzymatic error correction methods are cost-effective means to ameliorate errors in gene synthesis. Previous analyses of these methods relied on cloning and Sanger sequencing to evaluate their efficiencies, limiting quantitative assessment. Here, we develop a method to quantify errors in synthetic DNA by next-generation sequencing. We analyzed errors in model gene assemblies and systematically compared six different error correction enzymes across 11 conditions. We find that ErrASE and T7 Endonuclease I are the most effective at decreasing average error rates (up to 5.8-fold relative to the input), whereas MutS is the best for increasing the number of perfect assemblies (up to 25.2-fold). We are able to quantify differential specificities such as ErrASE preferentially corrects C/G transversions whereas T7 Endonuclease I preferentially corrects A/T transversions. More generally, this experimental and computational pipeline is a fast, scalable and extensible way to analyze errors in gene assemblies, to profile error correction methods, and to benchmark DNA synthesis methods.

Infection by Leishmania (Viannia) panamensis, the predominant etiologic agent for cutaneous leishmaniasis in Colombia, is characterized by a chronic mixed inflammatory response. Current treatment options are plagued by toxicity, lengthy treatment regimens, and growing evidence of drug resistance. Immunotherapy, modulating the immune system to mount a protective response, may provide an alternate therapeutic approach. We investigated the ability of the Toll-like receptor 9 (TLR9) ligand CpG to modulate established disease in the L (V) panamensis mouse model. Treatment of established infection with a high dose (50 μg) of CpG ameliorated disease and lowered parasite burden. Interestingly, immediately after treatment there was a significant increase in transforming growth factor β (TGF-β) and concomitantly an increase in T regulatory cell (Treg) function. Although a general reduction in cell-mediated immune cytokine and chemokine (gamma interferon [IFN-γ], interleukin 10 [IL-10], IL-13, IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-4, and MIP-1α) responses of the treated mice was observed, certain chemokines (RANTES, monocyte chemoattractant protein 1[MCP-1], and IP-10) were increased. Further, in peripheral blood mononuclear cells (PBMCs) from patients with cutaneous leishmaniasis, CpG treatment similarly exhibited a dose-response effect on the production of IFN-γ, IL-17, IL-10, and IL-13, with reductions observed at higher doses. To further understand the underlying mechanisms and cell populations driving the CpG mediated response, we examined the ex vivo dose effects mediated by the TLR9+ cell populations (dendritic cells, macrophages, and B cells) found to accumulate labeled CpG in vivo Notably, B cells altered the production of IL-17, IL-13, and IFN-γ, supporting a role for B cells functioning as antigen-presenting cells (APCs) and/or regulatory cells during infection. Interestingly, B cells have been previously demonstrated as a primary type of APC in patients infected with L (V) panamensis and thus may be useful targets of immunotherapy. Collectively, our results show that CpG-induced immune regulation leads to a dampening of the host immune response and healing in the mouse model, and it may provide an alternate approach to treatment of cutaneous leishmaniasis caused by L (V) panamensis.

Hyperalgesic priming, a model of pain chronification in the rat, is mediated by ryanodine receptor-dependent calcium release. Although ryanodine induces priming in both sexes, females are 5 orders of magnitude more sensitive, by an estrogen receptor α (EsRα)-dependent mechanism. An inositol 1,4,5-triphosphate (IP3) receptor inhibitor prevented the induction of priming by ryanodine. For IP3 induced priming, females were also more sensitive. IP3-induced priming was prevented by pretreatment with inhibitors of the sarcoendoplasmic reticulum calcium ATPase and ryanodine receptor. Antisense to EsRα prevented the induction of priming by low-dose IP3 in females. The induction of priming by an EsRα agonist was ryanodine receptor-dependent and prevented by the IP3 antagonist. Thus, an EsRα-dependent bidirectional interaction between endoplasmic reticulum IP3 and ryanodine receptor-mediated calcium signaling is present in the induction of hyperalgesic priming, in females. In cultured male DRG neurons, IP3 (100 μm) potentiated depolarization-induced transients produced by extracellular application of high-potassium solution (20 mm, K20), in nociceptors incubated with β-estradiol. This potentiation of depolarization-induced calcium transients was blocked by the IP3 antagonist, and not observed in the absence of IP3 IP3 potentiation was also blocked by ryanodine receptor antagonist. The application of ryanodine (2 nm), instead of IP3, also potentiated K20-induced calcium transients in the presence of β-estradiol, in an IP3 receptor-dependent manner. Our results point to an EsRα-dependent, reciprocal interaction between IP3 and ryanodine receptors that contributes to sex differences in hyperalgesic priming.SIGNIFICANCE STATEMENT The present study demonstrates a mechanism that plays a role in the marked sexual dimorphism observed in a model of the transition to chronic pain, hyperalgesic priming. This mechanism involves a reciprocal interaction between the endoplasmic reticulum receptors, IP3 and ryanodine, in the induction of priming, regulated by estrogen receptor α in the nociceptor of female rats. The presence of this signaling pathway modulating the susceptibility of nociceptors to develop plasticity may contribute to our understanding of sex differences observed clinically in chronic pain syndromes.

Raman nanoparticle probes are a potent class of optical labels for the interrogation of pathological and physiological processes in cells, bioassays, and tissues. Herein, we review the recent advancements in fluorescent and Raman imaging using oligodeoxyribonucleotide (ODN)-based nanoparticles and nanostructures, which show promise as effective tools for live-cell analysis. These nanodevices can be used to investigate a vast number of biological processes occurring at various levels, starting from those involving organelles, cells, tissues, and whole living organisms. ODN-based fluorescent and Raman probes have contributed to the achievement of significant advancements in the comprehension of the role played by specific analytes in pathological processes and have inaugurated new possibilities for diagnosing health conditions. The technological implications that have emerged from the studies herein described could open new avenues for innovative diagnostics aimed at identifying socially relevant diseases like cancer through the utilization of intracellular markers and/or guide surgical procedures based on fluorescent or Raman imaging. Particularly complex probe structures have been developed within the past five years, creating a versatile toolbox for live-cell analysis, with each tool possessing its own strengths and limitations for specific studies. Analyzing the literature reports in the field, we predict that the development of ODN-based fluorescent and Raman probes will continue in the near future, disclosing novel ideas on their application in therapeutic and diagnostic strategies.

We performed a phase I study of GTI-2040, an antisense oligonucleotide against ribonucleotide reductase mRNA, on a novel dosing schedule of days 1-4 and 15-18 by continuous infusion to examine efficacy and tolerability in patients with leukemia. A dose of 11 mg/kg/d was safely reached. Dose-limiting toxicities (DLTs) at the higher levels included elevated troponin I and liver function enzymes. There were no objective responses to GTI-2040 in this study; 7/24 patients were able to complete the predetermined three infusion cycles. Pharmacokinetic and pharmacodynamic studies were performed, indicating a trend towards increasing intracellular drug levels and decreasing RRM2 gene expression with increasing doses. This dose schedule may be considered if appropriate combinations are identified in preclinical studies.

PurposePrevious studies demonstrate that intratumoral CpG immunotherapy in combination with radiotherapy acts as an in-situ vaccine inducing antitumor immune responses capable of eradicating systemic disease. Unfortunately, most patients fail to respond. We hypothesized that immunotherapy can paradoxically upregulate immunosuppressive pathways, a phenomenon we term "rebound immune suppression," limiting clinical responses. We further hypothesized that the immunosuppressive enzyme indolamine-2,3-dioxygenase (IDO) is a mechanism of rebound immune suppression and that IDO blockade would improve immunotherapy efficacy.Experimental designWe examined the efficacy and immunologic effects of a novel triple therapy consisting of local radiotherapy, intratumoral CpG, and systemic IDO blockade in murine models and a pilot canine clinical trial.ResultsIn murine models, we observed marked increase in intratumoral IDO expression after treatment with radiotherapy, CpG, or other immunotherapies. The addition of IDO blockade to radiotherapy + CpG decreased IDO activity, reduced tumor growth, and reduced immunosuppressive factors, such as regulatory T cells in the tumor microenvironment. This triple combination induced systemic antitumor effects, decreasing metastases, and improving survival in a CD8(+) T-cell-dependent manner. We evaluated this novel triple therapy in a canine clinical trial, because spontaneous canine malignancies closely reflect human cancer. Mirroring our mouse studies, the therapy was well tolerated, reduced intratumoral immunosuppression, and induced robust systemic antitumor effects.ConclusionsThese results suggest that IDO maintains immune suppression in the tumor after therapy, and IDO blockade promotes a local antitumor immune response with systemic consequences. The efficacy and limited toxicity of this strategy are attractive for clinical translation. Clin Cancer Res; 22(17); 4328-40. ©2016 AACR.

The alternatively spliced products of LMNA, lamin C and prelamin A (the precursor to lamin A), are produced in similar amounts in most tissues and have largely redundant functions. This redundancy suggests that diseases, such as Hutchinson-Gilford progeria syndrome (HGPS), that are caused by prelamin A-specific mutations could be treated by shifting the output of LMNA more toward lamin C. Here, we investigated mechanisms that regulate LMNA mRNA alternative splicing and assessed the feasibility of reducing prelamin A expression in vivo. We identified an exon 11 antisense oligonucleotide (ASO) that increased lamin C production at the expense of prelamin A when transfected into mouse and human fibroblasts. The same ASO also reduced the expression of progerin, the mutant prelamin A protein in HGPS, in fibroblasts derived from patients with HGPS. Mechanistic studies revealed that the exon 11 sequences contain binding sites for serine/arginine-rich splicing factor 2 (SRSF2), and SRSF2 knockdown lowered lamin A production in cells and in murine tissues. Moreover, administration of the exon 11 ASO reduced lamin A expression in wild-type mice and progerin expression in an HGPS mouse model. Together, these studies identify ASO-mediated reduction of prelamin A as a potential strategy to treat prelamin A-specific diseases.

Background: Excessive inflammation in the periodontal tissue after tooth replantation can lead to inflammatory root resorption and interrupt periodontal tissue regeneration. We tested the hypothesis that nuclear factor-κB decoy oligodeoxynucleotide-loaded poly lactic-co-glycolic acid nanospheres (NF-PLGA) inhibit excessive inflammation and promote healing of periodontal tissue after replantation in rats.Methods: The upper right incisors of rats were extracted, immersed in different specific solutions, and replanted. The rats were euthanized at 7, 14, and 28 days after replantation. Morphological evaluation with micro-CT and histological assessment with hematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP) staining was performed. Additionally, we examined the expression of interleukin (IL)-1β, IL-6, transforming growth factor-β1 (TGF-β1), and fibroblast growth factor-2 (FGF-2) in the periodontal ligament (PDL) by performing immunohistological assessment.Results: The NF-PLGA group showed significantly greater dental root thickness than the other experimental groups. Root resorption was not observed after the application of NF-PLGA on day 7. The application of NF-PLGA also resulted in a significantly lower number of TRAP-positive osteoclasts on days 7 and 14 after replantation. Significantly lower expression of IL-1β and IL-6 and higher expression of TGF-β1 and FGF-2 were observed under the application of NF-PLGA in the PDL.Conclusions: NF-PLGA promoted the healing process by inhibiting the initial excessive inflammatory response in the PDL, preventing root resorption, and promoting periodontal tissue regeneration. The findings also suggested that the PLGA nanospheres-mediated transfection of the decoy oligodeoxynucleotides can be useful for the clinical application of replanted tooth root surfaces. [ABSTRACT FROM AUTHOR]

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules consisting of two ligands joined by a linker, enabling them to simultaneously bind with an E3 ligase and a protein of interest (POI) and trigger proteasomal degradation of the POI. Limitations of PROTAC include lack of potent E3 ligands, poor cell selectivity, and low permeability. AS1411 is an antitumor aptamer specifically recognizing a membrane-nucleus shuttling nucleolin (NCL). Here, we repurpose AS1411 as a ligand for an E3 ligase mouse double minute 2 homolog (MDM2) via anchoring the NCL-MDM2 complex. Then, we construct an AS1411-NCL-MDM2-based PROTAC (ANM-PROTAC) by conjugating AS1411 with large-molecular-weight ligands for "undruggable" oncogenic STAT3, c-Myc, p53-R175H, and AR-V7. We show that the ANM-PROTAC efficiently penetrates tumor cells, recruits MDM2 and degrades the POIs. The ANM-PROTAC achieves tumor-selective distribution and exhibits excellent antitumor activity with no systemic toxicity. This is a PROTAC with built-in tumor-targeting and cell-penetrating capacities., Competing Interests: Declaration of interests Y.L. has patent application related to this work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hepatocyte growth factor receptor (c-Met) is a suitable molecular target for the targeted therapy of cancer. Novel c-Met-targeting drugs need to be developed because conventional small-molecule inhibitors and antibodies of c-Met have some limitations. To synthesize such drugs, we developed a bispecific DNA nanoconnector (STPA) to inhibit c-Met function. STPA was constructed by using DNA triangular prism as a scaffold and aptamers as binding molecules. After c-Met-specific SL1 and nucleolin-specific AS1411 aptamers were integrated with STPA, STPA could bind to c-Met and nucleolin on the cell membrane. This led to the formation of the c-Met/STPA/nucleolin complex, which in turn blocked c-Met activation. In vitro experiments showed that STPA could not only inhibit the c-Met signaling pathways but also facilitate c-Met degradation through lysosomes. STPA also inhibited c-Met-promoted cell migration, invasion, and proliferation. The results of in vivo experiments showed that STPA could specifically target to tumor site in xenograft mouse model, and inhibit tumor growth with low toxicity by downregulating c-Met pathways. This study provided a novel and simple strategy to develop c-Met-targeting drugs for the targeted therapy of cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Here, a novel targeted nanostructure complex was designed as an alternative to the traditional treatment approaches for breast cancer. A delivery system utilizing CuS nanoparticles (CuS NPs) was developed for the purpose of targeted administration of doxorubicin (Dox), an anticancer agent. To regulate Dox release, chitosan (CS), a biodegradable and hydrophilic polymer with biocompatible properties, was applied to coat the Dox-loaded CuS NPs. Furthermore, AS1411 aptamer, served as a targeting agent for breast cancer cells (MCF-7 and 4T1 cells), was conjugated with CS-Dox-CuS NPs effectively. To assess the effectiveness of APT-CS-CuS NPs, various methods such as flow cytometry analysis, MTT assay, fluorescence imaging, and in vivo antitumor efficacy were employed. The hollow core and porous surface of CuS NPs improved the Dox loading capacity and entrapment efficiency (almost 100%). The rate of drug release at the tumor site (citrate buffer with pH 5.6) exhibited a marked increase in comparison to that observed within the physiological environment (phosphate buffer with pH 7.4). The targeted formulation (APT-CS-Dox-CuS NPs) significantly increased cytotoxicity of the Dox payload in target cells, including 4T1 (p ≤ 0.0001 (****)) and MCF7 (p ≤ 0.01 (**)) cells compared to CHO cells. Moreover, the ability of tumor growth inhibition of the targeted system was significantly (p ≤ 0.05 (*)) more than free Dox in tumor-bearing mice. The findings indicate that the targeted formulation augmented effectiveness and specificity while minimizing harm to non-targeted cells, signifying its potential as a sophisticated cancer drug delivery system., Competing Interests: Declaration of competing interest There is no conflict of interest about this article., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Targeted delivery of self-antigens to the immune system in a mode that stimulates a tolerance-inducing pathway has proven difficult. To address this hurdle, we developed a vaccine based-approach comprised of two synthetic controlled-release biomaterials, poly(lactide-co-glycolide; PLGA) microparticles (MPs) encapsulating denatured insulin (key self-antigen in type 1 diabetes; T1D), and PuraMatrix(TM) peptide hydrogel containing granulocyte macrophage colony-stimulating factor (GM-CSF) and CpG ODN1826 (CpG), which were included as vaccine adjuvants to recruit and activate immune cells. Although CpG is normally considered pro-inflammatory, it also has anti-inflammatory effects, including enhancing IL-10 production. Three subcutaneous administrations of this hydrogel (GM-CSF/CpG)/insulin-MP vaccine protected 40% of NOD mice from T1D. In contrast, all control mice became diabetic. In vitro studies indicate CpG stimulation increased IL-10 production, as a potential mechanism. Multiple subcutaneous injections of the insulin containing formulation resulted in formation of granulomas, which resolved by 28 weeks. Histological analysis of these granulomas indicated infiltration of a diverse cadre of immune cells, with characteristics reminiscent of a tertiary lymphoid organ, suggesting the creation of a microenvironment to recruit and educate immune cells. These results demonstrate the feasibility of this injectable hydrogel/MP based vaccine system to prevent T1D.

Hyperalgesic priming, a form of neuroplasticity in nociceptors, is a model of the transition from acute to chronic pain in the rat, which involves signaling from the site of an acute tissue insult in the vicinity of the peripheral terminal of a nociceptor to its cell body that, in turn, induces a signal that travels back to the terminal to mediate a marked prolongation of prostaglandin E2-induced hyperalgesia. In the present experiments, we studied the underlying mechanisms in the cell body and compared them to the mechanisms in the nerve terminal. Injection of a cell-permeant cAMP analog, 8-bromo cAMP, into the dorsal root ganglion induced mechanical hyperalgesia and priming with an onset more rapid than when induced at the peripheral terminal. Priming induced by intraganglion 8-bromo cAMP was prevented by an oligodeoxynucleotide antisense to mRNA for a transcription factor, cAMP response element-binding protein (CREB), and by an inhibitor of importin, which is required for activated CREB to get into the nucleus. While peripheral administration of 8-bromo cAMP also produced hyperalgesia, it did not produce priming. Conversely, interventions administered in the vicinity of the peripheral terminal of the nociceptor that induces priming-PKCε activator, NGF, and TNF-α-when injected into the ganglion produce hyperalgesia but not priming. The protein translation inhibitor cordycepin, injected at the peripheral terminal but not into the ganglion, reverses priming induced at either the ganglion or peripheral terminal of the nociceptor. These data implicate different mechanisms in the soma and terminal in the transition to chronic pain.

Cathelicidins are a gene family best known for their antimicrobial action, but the diverse mature peptides they encode also have other host defense functions. The human cathelicidin peptide LL-37 enhances recognition of nucleic acids, an action whose significance is seen in human diseases such as psoriasis where it is associated with increased type 1 IFN production. This function has been attributed to the extracellular action of the peptide to facilitate uptake of nucleic acids. In this study, we demonstrate that the murine mature cathelicidin peptide (CRAMP), encoded by the mouse gene (Camp), is functionally distinct from the human mature peptide (LL-37), as it does not facilitate CpG entry. However, mouse cathelicidin does influence recognition of CpG as bone marrow-derived dendritic cells from Camp(-/-) mice have impaired CpG responses and Camp(-/-) mice had impaired response to CpG given i.v. or s.c. We show that cathelicidin concentrates in Lamp1 positive compartments, is colocalized with CpG in the endolysosome, can be immunoprecipitated with TLR9, and binds to CpG intracellulary. Collectively, these results indicate that the functions of cathelicidin in control of TLR9 activation may include both intracellular and extracellular effects.

Dendritic DNA molecules, referred to as DNA dendrons, consist of multiple covalently linked strands and are expected to improve the cellular uptake and potency of therapeutic oligonucleotides because of their multivalency. In this study, we developed an efficient synthetic method for producing DNA dendrons using strain-promoted azide-alkyne cycloaddition. Integration of the antitumor aptamer AS1411 into DNA dendrons enhanced cellular uptake and antiproliferative activity in cancer cells. These findings demonstrate that the incorporation of multivalent aptamers into DNA dendrons can effectively boost their therapeutic effects.

Conventional chemotherapy and poor targeted delivery in brain cancer resulting to poor treatment and develop resistance to anticancer drugs. Meanwhile, it is quite challenging to diagnose/detection of brain tumor at early stage of cancer which resulting in severity of the disease. Despite extensive research, effective treatment with real-time imaging still remains completely unavailable, yet. In this study, two brain cancer cell specific moieties i.e., AS1411 aptamer and RGD are decorated on the surface of chitosan-PLGA nanoparticles to improve targeted co-delivery of docetaxel (DTX) and upconversion nanoparticles (UCNP) for effective brain tumor therapy and real-time imaging. The nanoparticles were developed by a slightly modified emulsion/solvent evaporation method. This investigation also translates the successful synthesis of TPGS-chitosan, TPGS-RGD and TPGS-AS1411 aptamer conjugates for making PLGA nanoparticle as a potential tool of the targeted co-delivery of DTX and UCNP to the brain cancer cells. The developed nanoparticles have shown an average particle size <200 nm, spherical in shape, high encapsulation of DTX and UCNP in the core of nanoparticles, and sustained release of DTX up to 72 h in phosphate buffer saline (pH 7.4). AS1411 aptamer and RGD functionalized theranostic chitosan-PLGA nanoparticles containing DTX and UCNP (DUCPN-RGD-AS1411) have achieved greater cellular uptake, 89-fold improved cytotoxicity, enhanced cancer cell arrest even at lower drug conc., improved bioavailability with higher mean residence time of DTX in systemic circulation and brain tissues. Moreover, DUCPN-RGD-AS1411 have greatly facilitated cellular internalization and higher accumulation of UCNP in brain tissues. Additionally, DUCPN-RGD-AS1411 demonstrated a significant suppression in tumor growth in brain-tumor bearing xenograft BALB/c nude mice with no impressive sign of toxicities. DUCPN-RGD-AS1411 has great potential to be utilized as an effective and safe theranostic tool for brain cancer and other life-threatening cancer therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

CpG oligodeoxynucleotides (ODNs), as a novel type of adjuvant with immunomodulatory effects, are recognized by Toll-like receptors (TLRs) in Litopenaeus vannamei. In the present study, eleven LvTLRs-pCMV recombinants (rLvTLRs) were constructed to investigate the relationships between various CpG ODNs and different LvTLRs in human embryonic kidney 293T (HEK293T) cells, which was further confirmed by bio-layer interferometry (BLI) technique. The results of dual luciferase reporter assay showed that every LvTLR could activate multiple downstream genes, mainly including NF-κB, CREB, ISRE, IL-6-promoter, TNF-α-promoter and Myc, thereby inducing main signaling pathways in shrimps. Most CpG ODNs possessed affinities to more than one LvTLR, while each LvTLR could recognize multiple CpG ODNs, and the widely recognized ligands within CpG ODNs are A-class and B-class. Moreover, BLI analysis showed that CpG 2216, Cpg 2006, CpG 2143 and CpG 21425 exhibited dose-dependent affinity to the expressed TLR protein, which were consistent with the results in HEK293T cells. It suggested that the interactions of CpG ODNs with LvTLRs were indispensable for the immune regulation triggered by CpG ODNs, and these findings would lay foundations for studying the activations of LvTLRs to immune signaling pathways and shedding lights on the immune functions and mechanisms of CpG ODNs., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Due to its inherent membrane structure, a nanostructure enveloped by an active cell membrane possesses distinctive characteristics such as prolonged presence in the bloodstream, precise identification capabilities, and evasion of immune responses. This research involved the production of biomimetic nanoparticles, specifically hollow gold nanoparticles (HGNPs) loaded with methotrexate (MTX), which were further coated with cancer cell membrane. These nanoparticles were then adorned with AS1411 aptamer to serve as a targeting agent (Apt-CCM-HG@MTX). The nanoplatform demonstrated precise targeting towards cancer cells due to its dual-targeting characteristic (AS1411 aptamer and C26 cancer cell membrane), exhibiting uniformity in distribution. It also displayed a desirable response to photothermal stimulation, controlled release of drugs, and exceptional properties for fluorescence imaging. The system was composed of spherical HGNPs measuring 51.33 ± 5.70 nm in diameter, which were effectively loaded with MTX using a physical absorption method. The encapsulation efficiency achieved was recorded at 79.54 %, while the loading efficiency reached 38.21 %. The targeted formulation demonstrated a noteworthy mortality of approximately 45 % in the nucleolin positive cell line, C26, as determined by in vitro cytotoxicity assays. As a result of the functionalization process applied to the homologous binding adhesion molecules found in cancer cell membranes and targeting ability of AS1411 aptamer, Apt-CCM-HG@MTX demonstrated a substantial enhancement in targeting tumors and facilitating cellular uptake during in vivo experiments. Furthermore, under NIR radiation the photothermal effect exhibited by Apt-CCM-HG@MTX in the tumor area was notably robust due to the distinctive attributes of HGNPs. The conclusions obtained from this study have the potential to assist in adopting a bioinspired strategy that will significantly improve the effective management of MTX and therapy for individuals with colorectal cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Adjuvants are critical components for vaccines, which enhance the strength and longevity of the antibody response and influence the types of immune response. Limited research has been conducted on the immunogenicity and protective efficacy of various adjuvants in malaria transmission-blocking vaccines (TBVs). In this study, we formulated a promising TBV candidate antigen, the P. berghei ookinete surface antigen PSOP25, with different types of adjuvants, including the TLR4 agonist monophosphoryl lipid A (MPLA), the TLR9 agonist cytosine phosphoguanosine oligodeoxynucleotides (CpG ODN 1826) (CpG), a saponin adjuvant QS-21, aluminum hydroxide (Alum), and two combination adjuvants MPLA + QS-21 and QS-21 + CpG. We demonstrated that adjuvanted vaccines results in elevated elicited antibody levels, increased proliferation of plasma cells, and efficient formation of germinal centers (GCs), leading to enhanced long-term protective immune responses. Furthermore, CpG group exhibited the most potent inhibition of ookinete formation and transmission-blocking activity. We found that the rPSOP25 with CpG adjuvant was more effective than MPLA, QS-21, MPLA + QS-21, QS-21 + CpG adjuvants in dendritic cells (DCs) activation and differentiation. Additionally, the CpG adjuvant elicited more rubust immune memory response than Alum adjuvant. CpG and QS-21 adjuvants could activate the Th1 response and promote the secretion of IFN-γ and TNF-α. PSOP25 induced a higher number of Tfh cells in splenocytes when combined with MPLA, CpG, and QS-21 + CpG; and there was no increase in these cell populations when PSOP25 was administered with Alum. In conclusion, CpG may confer enhanced efficacy for the rPSOP25 vaccine, as evidenced by the ability of the elicited antisera to induce protective immune responses and improved transmission-blocking activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Enterovirus D68 (EV-D68), a pathogen that causes respiratory symptoms, mainly in children, has been implicated in acute flaccid myelitis, which is a poliomyelitis-like paralysis. Currently, there are no licensed vaccines or treatments for EV-D68 infections. Here, we investigated the optimal viral inactivation reagents, vaccine adjuvants, and route of vaccination in mice to optimize an inactivated whole-virion (WV) vaccine against EV-D68. We used formalin, β-propiolactone (BPL), and hydrogen peroxide as viral inactivation reagents and compared their effects on antibody responses. Use of any of these three viral inactivation reagents effectively induced neutralizing antibodies. Moreover, the antibody response induced by the BPL-inactivated WV vaccine was enhanced when adjuvanted with cytosine phosphoguanine oligodeoxynucleotide (CpG ODN) or AddaVax (MF59-like adjuvant), but not with aluminum hydroxide (alum). Consistent with the antibody response results, the protective effect of the inactivated WV vaccine against the EV-D68 challenge was enhanced when adjuvanted with CpG ODN or AddaVax, but not with alum. Further, while the intranasal inactivated WV vaccine induced EV-D68-specific IgA antibodies in the respiratory tract, it was less protective against EV-D68 challenge than the injectable vaccine. Thus, an injectable inactivated EV-D68 WV vaccine prepared with appropriate viral inactivation reagents and an optimal adjuvant is a promising EV-D68 vaccine., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yasuo Yoshioka reports financial support was provided by The Research Foundation for Microbial Diseases of Osaka University. Chikako Kataoka-Nakamura reports financial support was provided by The Research Foundation for Microbial Diseases of Osaka University. Yuta Kunishima reports financial support was provided by The Research Foundation for Microbial Diseases of Osaka University. Yasuo Yoshioka reports a relationship with The Research Foundation for Microbial Diseases of Osaka University that includes: employment. Chikako Kataoka-Nakamura reports a relationship with The Research Foundation for Microbial Diseases of Osaka University that includes: employment. Yuta Kunishima reports a relationship with The Research Foundation for Microbial Diseases of Osaka University that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A successful vaccine depends on its capacity to elicit a protective immune response against the target pathogen. The adjuvant used plays an important role in enhancing and directing the immune response. Liposomes are vaccine adjuvants that allow the co-encapsulation of antigens and immunostimulants. Our aim was to evaluate the adjuvanticity of a cationic liposome (Lip) formulated with a novel gemini lipopeptide (AG2-C16) alone or in combination with CpG-ODN as immunostimulants. To achieve this, we used the recombinant clumping factor of Staphylococcus aureus (rClfA) as a model antigen, in a murine model. We characterized the formulations by DLS, Cryo-SEM, and TEM, and analyzed the humoral and cellular immune responses induced in BALB/c and C57BL/6J mice injected with free rClfA and three formulations: Lip + CpG-ODN + rClfA, Lip + AG2-C16 + rClfA and Lip + AG2-C16 + CpG-ODN + rClfA. The addition of immunostimulants to the liposomes did not change the membrane diameter but affected their hydrodynamic diameter, z-potential, and homogeneity. All liposomal formulations were able to stimulate a specific humoral response, with high serum IgG, IgG1 and IgG2a or IgG2c titers in BALB/c or C57BL/6J mice, respectively. In addition, increased vaginal IgG levels were detected after injection, with no specific IgA. The cellular immunity induced by Lip + AG2-C16 + CpG-ODN + rClfA was characterized by a predominant Th1 profile, with the co-induction of Th2 and Th17 cells, and IFN-γ + cytotoxic T cells. Furthermore, we studied the capacity of the different formulations to stimulate murine keratinocytes and fibroblasts in vitro. While no formulation activated keratinocytes, Lip + AG2-C16 + CpG-ODN increased the expression of CXCL9 in fibroblasts. These results suggest Lip + AG2-C16 + CpG-ODN as a promising adjuvant candidate to be used in vaccines against pathogens that require Th1/Th2/Th17 combined profiles, like S. aureus. Additionally, based on the IFN-γ + cytotoxic T cells stimulation and the CXCL9 production by fibroblasts, we propose the use of this adjuvant formulation for the stimulation of a Th1 profile., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Carolina Veaute reports travel was provided by ECOS-Sud. Ivana Reidel reports travel was provided by ECOS-Sud. Jean-Francois Jegou reports travel was provided by ECOS-Sud. Carolina Veaute has patent ##20180103649 (INPI Argentina) pending to Licensee. Ivana Reidel has patent ##20180103649 (INPI Argentina) pending to Licensee. Maria Ines Garcia has patent ##20180103649 (INPI Argentina) pending to Licensee. Diana Muller has patent ##20180103649 (INPI Argentina) pending to Licensee., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Herpesvirus of turkey (HVT) increases activation of T cells in 1-day-old chickens when administered in ovo . This study evaluated whether adding cytosine-guanosine oligodeoxynucleotides (CpG ODNs) to the HVT vaccine could enhance the adjuvant effect of HVT. We used a CpG ODN dose of 10  μ g per egg. The experimental groups were (1) diluent-only control (sham), (2) HVT, (3) HVT+CpG ODN, (4) HVT+non-CpG ODN, (5) CpG ODN, and (6) non-CpG ODN control. Cellular response evaluation included measuring the frequencies of macrophages (KUL01 + MHC-II + ), gamma delta T cells ( γδ TCR + MHC-II + ), CD4 + , and CD8 + T cell subsets, including double-positive (DP) cells. In addition, CD4 + and CD8 + T cell activation was evaluated by measuring the cellular expression of major histocompatibility complex class II (MHC-II), CD44 or CD28 costimulatory molecules. An adjuvant effect was considered when HVT+CpG ODN, but not HVT+non CpG ODN, or CpG ODN, or non-CpG ODN, induced significantly increased effects on any of the immune parameters examined when compared with HVT. The findings showed that (1) HVT vaccination induced significantly higher frequencies of γδ + MHC-II + and CD4 + CD28 + T cells when compared with sham chickens. Frequencies of DP and CD4 + CD28 + T cells in HVT-administered birds were significantly higher than those observed in the non-CpG ODN group. (2) Groups receiving HVT+CpG ODN or CpG ODN alone were found to have significantly increased frequencies of activated CD4 + and CD8 + T cells, when compared with HVT. Our results show that CpG ODN administration in ovo with or without HVT significantly increased frequencies of activated CD4 + and CD8 + T cells.

Introduction: Therapeutic vaccination in tuberculosis (TB) represents a Host Directed Therapy strategy which enhances immune responses in order to improve clinical outcomes and shorten TB treatment. Previously, we have shown that the subunit H56:IC31 vaccine induced both humoral and cellular immune responses when administered to TB patients adjunctive to standard TB treatment (TBCOX2 study, NCT02503839). Here we present the longitudinal whole blood gene expression patterns in H56:IC31 vaccinated TB patients compared to controls receiving standard TB treatment only., Methods: The H56:IC31 group (N=11) and Control group (N=7) underwent first-line TB treatment for 182 days. The H56:IC31 group received 5 micrograms of the H56:IC31 vaccine (Statens Serum Institut; SSI, Valneva Austria GmbH) intramuscularly at day 84 and day 140. Total RNA was extracted from whole blood samples collected in PAXgene tubes on days 0, 84, 98, 140, 154, 182 and 238. The expression level of 183 immune-related genes was measured by high-throughput microfluidic qPCR (Biomark HD system, Standard BioTools)., Results: The targeted gene expression profiling unveiled the upregulation of modules such as interferon (IFN) signalling genes, pattern recognition receptors and small nucleotide guanosine triphosphate (GTP)-ases in the vaccinated group compared to controls two weeks after administration of the first H56:IC31 vaccine. Additionally, the longitudinal analysis of the Adolescent Cohort Study-Correlation of Risk (ACS-COR) signature showed a progressive downregulation in both study arms towards the end of TB treatment, in congruence with reported treatment responses and clinical improvements. Still, two months after the end of TB treatment, vaccinated patients, and especially those developing both cellular and humoral vaccine responses, showed a lower expression of the ACS-COR genes compared to controls., Discussion: Our data report gene expression patterns following H56:IC31 vaccination which might be interpreted as a lower risk of relapse in therapeutically vaccinated patients. Further studies are needed to conclude if these gene expression patterns could be used as prognostic biosignatures for therapeutic TB vaccine responses., Competing Interests: RM is employee at SSI that developed the H56:IC31 vaccine. He is not inventor of related patents and have no personal financial interests. IC31 is a trademark of Valneva Austria GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Alonso-Rodríguez, Vianello, van Veen, Jenum, Tonby, van Riessen, Lai, Mortensen, Ottenhoff and Dyrhol-Riise.)

The innate immune response relies on the ability of host cells to rapidly detect and respond to microbial nucleic acids. Toll-like receptors (TLRs), a class of pattern recognition receptors (PRRs), play a fundamental role in distinguishing self from non-self at the molecular level. In this study, we focused on TLR21, an avian TLR that recognizes DNA motifs commonly found in bacterial genomic DNA, specifically unmethylated CpG motifs. TLR21 is believed to act as a functional homologue to mammalian TLR9. By analysing TLR21 signalling in chickens, we sought to elucidate avian TLR21 activation outputs in parallel to that of other nucleic acid species. Our analyses revealed that chicken TLR21 (chTLR21) triggers the activation of NF-κB and induces a potent type-I interferon response in chicken macrophages, similar to the signalling cascades observed in mammalian TLR9 activation. Notably, the transcription of interferon beta (IFNB) by chTLR21 was found to be dependent on both NF-κB and IRF7 signalling, but independent of the TBK1 kinase, a distinctive feature of mammalian TLR9 signalling. These findings highlight the conservation of critical signalling components and downstream responses between avian TLR21 and mammalian TLR9, despite their divergent evolutionary origins. These insights into the evolutionarily conserved mechanisms of nucleic acid sensing contribute to the broader understanding of host-pathogen interactions across species., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

CpG oligodeoxynucleotides (CpG ODNs) boost the humoral and cellular immune responses to antigens through interaction with Toll-like receptor 9 (TLR9). These CpG ODNs have been extensively utilized in human vaccines. In our study, we evaluated five B-type CpG ODNs that have stimulatory effects on pigs by measuring the proliferation of porcine peripheral blood mononuclear cells (PBMCs) and assessing interferon gamma (IFN-γ) secretion. Furthermore, this study examined the immunoenhancing effects of the MF59 and CpG ODNs compound adjuvant in mouse and piglet models of porcine epidemic diarrhea virus (PEDV) subunit vaccine administration. The in vitro screening revealed that the CpG ODN named CpG5 significantly stimulated the proliferation of porcine PBMCs and elevated IFN-γ secretion levels. In the mouse vaccination model, CpG5 compound adjuvant significantly bolstered the humoral and cellular immune responses to the PEDV subunit vaccines, leading to Th1 immune responses characterized by increased IFN-γ and IgG2a levels. In piglets, the neutralizing antibody titer was significantly enhanced with CpG5 compound adjuvant, alongside a considerable increase in CD8+ T lymphocytes proportion. The combination of MF59 adjuvant and CpG5 exhibits a synergistic effect, resulting in an earlier, more intense, and long-lasting immune response in subunit vaccines for PEDV. This combination holds significant promise as a robust candidate for the development of vaccine adjuvant., Competing Interests: Authors QP and LL were employed by Nanjing JSIAMA Biopharmaceuticals Ltd. Author YZ and ED were employed by Yangling Carey Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Liu, Li, Sun, Pan, Zheng, Liu, Zhao, Wang, Liu and Du.)