Learning Curve Effect of Combined Technique Thrombectomy as First-Line Attempt for Acute Ischemic Stroke: A Single-Center Retrospective Study.

OBJECTIVES: We retrospectively examined the initial experience and learning curve after the introduction of thrombectomy with the combined technique using an aspiration catheter and a stent retriever as first-line attempt for acute ischemic stroke. METHODS: Consecutive patients undergoing thrombectomy for acute ischemic stroke at our institution between January 2020 and December 2022 were divided into three groups according to the year of thrombectomy. Patient characteristics and procedural, safety, and clinical outcomes were compared between the three year periods to determine predictors of favorable clinical outcome. RESULTS: In 2020, 2021, and 2022, the numbers of patients were 74, 70, and 90, respectively, with similar patient characteristics across the three years; successful recanalization rates were 79.7%, 97.1%, and 93.3%, respectively (p<0.01 for the first 2 years); median procedure times were 67, 43, and 32 minutes, respectively (p<0.01 for the first 2 years and p=0.018 for the last 2 years); first pass effect rates were 20.3%, 41.4%, and 44.4%, respectively (p<0.01 for the first 2 years); symptomatic intracranial hemorrhage rates were 14.9%, 2.9%, and 1.1%, respectively (p=0.018 for the first 2 years); and percentages of modified Rankin Scale score 0 to 2 at 90 days were 24.3%, 42.9%, and 41.1%, respectively (p=0.022 for the first 2 years). Procedure time (p=0.038) and successful recanalization (p=0.041) were independent predictors of favorable clinical outcome. CONCLUSIONS: The learning curve effect of the combined technique may be associated with better clinical outcome due to increased successful recanalization rates, shortened procedure time, and reduced symptomatic intracranial hemorrhage.

Co-Delivery of a Novel Lipidated TLR7/8 Agonist and Hemagglutinin-Based Influenza Antigen Using Silica Nanoparticles Promotes Enhanced Immune Responses.

Co-delivery of antigens and adjuvants to the same antigen-presenting cells (APCs) can significantly improve the efficacy and safety profiles of vaccines. Here, we report amine-grafted silica nanoparticles (A-SNP) as a tunable vaccine co-delivery platform for TLR7/8 agonists along with the recombinant influenza antigen hemagglutinin H7 (H7) to APCs. A-SNP of two different sizes (50 and 200 nm) were prepared and coated with INI-4001 at different coating densities, followed by co-adsorption of H7. Both INI-4001 and H7 showed >90% adsorption to the tested A-SNP formulations. TNF-α and IFN-α cytokine release by human peripheral blood mononuclear cells as well as TNF-α, IL-6, and IL-12 release by mouse bone marrow-derived dendritic cells revealed that the potency of the INI-4001-adsorbed A-SNP (INI-4001/A-SNP) formulations was improved relative to aqueous formulation control. This improved potency was dependent on particle size and ligand coating density. In addition, slow-release profiles of INI-4001 were measured from INI-4001/A-SNP formulations in plasma with 30-50% INI-4001 released after 7 days. In vivo murine immunization studies demonstrated significantly improved H7-specific humoral and Th1/Th17-polarized T cell immune responses with no observed adverse reactions. Low-density 50 nm INI-4001/A-SNP elicited significantly higher IFN-γ and IL-17 induction over that of the H7 antigen-only group and INI-4001 aqueous formulation controls. In summary, this work introduces an effective and biocompatible SNP-based co-delivery platform that enhances the immunogenicity of TLR7/8 agonist-adjuvanted subunit influenza vaccines.

Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx.

Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca2+ elevation enhances EV release, we screened 80 hit compounds and identified compound 634 as a Ca2+ influx inducer. 634 enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from 634-treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca2+ elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of 634. The analogues that retained the ability to induce Ca2+ influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca2+ influx. The levels of Ca2+ induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, 634, enhances the release of EVs with immunostimulatory potency via induction of Ca2+ influx. This agent is a novel tool for EV-based immune studies and vaccine development.

Treatment of pancreatic cancer with irreversible electroporation and intratumoral CD40 antibody stimulates systemic immune responses that inhibit liver metastasis in an orthotopic model.

BACKGROUND: Pancreatic cancer (PC) has a poor prognosis, and most patients present with either locally advanced or distant metastatic disease. Irreversible electroporation (IRE) is a non-thermal method of ablation used clinically in locally advanced PC, but most patients eventually develop distant recurrence. We have previously shown that IRE alone is capable of generating protective, neoantigen-specific immunity. Here, we aim to generate meaningful therapeutic immune effects by combining IRE with local (intratumoral) delivery of a CD40 agonistic antibody (CD40Ab). METHODS: KPC46 organoids were generated from a tumor-bearing male KrasLSL-G12D-p53LSL-R172H-Pdx-1-Cre (KPC) mouse. Orthotopic tumors were established in the pancreatic tail of B6/129 F1J mice via laparotomy. Mice were randomized to treatment with either sham laparotomy, IRE alone, CD40Ab alone, or IRE followed immediately by CD40Ab injection. Metastatic disease and immune infiltration in the liver were analyzed 14 days postprocedure using flow cytometry and multiplex immunofluorescence imaging with spatial analysis. Candidate neoantigens were identified by mutanome profiling of tumor tissue for ex vivo functional analyses. RESULTS: The combination of IRE+CD40 Ab improved median survival to greater than 35 days, significantly longer than IRE (21 days) or CD40Ab (24 days) alone (p<0.01). CD40Ab decreased metastatic disease burden, with less disease in the combination group than in the sham group or IRE alone. Immunohistochemistry of liver metastases revealed a more than twofold higher infiltration of CD8+T cells in the IRE+CD40 Ab group than in any other group (p<0.01). Multiplex immunofluorescence imaging revealed a 4-6 fold increase in the density of CD80+CD11c+ activated dendritic cells (p<0.05), which were spatially distributed throughout the tumor unlike the sham group, where they were restricted to the periphery. In contrast, CD4+FoxP3+ T-regulatory cells (p<0.05) and Ly6G+myeloid derived cells (p<0.01) were reduced and restricted to the tumor periphery in the IRE+CD40 Ab group. T-cells from the IRE+CD40 Ab group recognized significantly more peptides representing candidate neoantigens than did T-cells from the IRE or untreated control groups. CONCLUSIONS: IRE can induce local tumor regression and neoantigen-specific immune responses. Addition of CD40Ab to IRE improved dendritic cell activation and neoantigen recognition, while generating a strong systemic antitumor T-cell response that inhibited metastatic disease progression.

Factors related to white thrombi in acute ischemic stroke in cancer patients.

OBJECTIVES: Thrombi in cerebral large vessel occlusion associated with active cancer are often fibrin and platelet-rich white thrombi. However, evaluating the thrombus composition in a short time before thrombectomy is often ineffective. We sought to determine factors related to white thrombi in acute ischemic stroke due to large vessel occlusion in cancer patients. METHODS: Consecutive cancer patients undergoing thrombectomy for acute ischemic stroke due to large vessel occlusion between January 2018 and May 2022 were retrospectively reviewed. The patients were classified into white thrombus and red thrombus groups on the basis of the pathological findings of retrieved thrombi. Patient characteristics and laboratory findings were compared between the two groups. RESULTS: There were 12 patients in the white thrombus group and 11 patients in the red thrombus group. Active cancer was significantly more in the white thrombus group than in the red thrombus group (91.7% vs. 36.3%, p = 0.0094). Internal carotid artery occlusion was significantly less in the white thrombus group than in the red thrombus group (0% vs. 36.4%, p = 0.037). Among laboratory findings, D-dimer levels were an independent factor associated with white thrombi (odds ratio 8.97 [95% confidence interval 1.71-368.99], p < 0.0001). The cutoff value of D-dimer levels for predicting white thrombi was 3.5 µg/mL (83.3% sensitivity and 100% specificity). CONCLUSIONS: In acute ischemic stroke in cancer patients, active cancer, no internal carotid artery occlusion, and higher D-dimer levels (≥3.5 µg/mL) may be associated with occlusion with fibrin and platelet-rich white thrombi.

A Dual Adjuvant System for Intranasal Boosting of Local and Systemic Immunity for Influenza Vaccination.

Systemically vaccinated individuals against COVID-19 and influenza may continue to support viral replication and shedding in the upper airways, contributing to the spread of infections. Thus, a vaccine regimen that enhances mucosal immunity in the respiratory mucosa is needed to prevent a pandemic. Intranasal/pulmonary (IN) vaccines can promote mucosal immunity by promoting IgA secretion at the infection site. Here, we demonstrate that an intramuscular (IM) priming-IN boosting regimen with an inactivated influenza A virus adjuvanted with the liposomal dual TLR4/7 adjuvant (Fos47) enhances systemic and local/mucosal immunity. The IN boosting with Fos47 (IN-Fos47) enhanced antigen-specific IgA secretion in the upper and lower respiratory tracts compared to the IM boosting with Fos47 (IM-Fos47). The secreted IgA induced by IN-Fos47 was also cross-reactive to multiple influenza virus strains. Antigen-specific tissue-resident memory T cells in the lung were increased after IN boosting with Fos47, indicating that IN-Fos47 established tissue-resident T cells. Furthermore, IN-Fos47 induced systemic cross-reactive IgG antibody titers comparable to those of IM-Fos47. Neither local nor systemic reactogenicity or adverse effects were observed after IN delivery of Fos47. Collectively, these results indicate that the IM/IN regimen with Fos47 is safe and provides both local and systemic anti-influenza immune responses.

Moyamoya Syndrome in a Patient with Williams Syndrome: A Case Report.

INTRODUCTION: Moyamoya syndrome associated with Williams syndrome is very rare but has been reported to have severe outcomes. Here, we reported a case of Williams syndrome with moyamoya syndrome that was confirmed by the presence of an RNF213 mutation. CASE PRESENTATION: A 6-year-old boy with Williams syndrome presented with right hemiparesis induced by hyperventilation. Magnetic resonance angiography and cerebral angiography showed severe stenosis of the bilateral internal carotid arteries and development of moyamoya vessels. Genetic analysis identified a heterozygous c.14576G>A (p.R4859K) mutation in RNF213. Moyamoya syndrome was diagnosed, and bilateral indirect revascularization surgery was conducted without complications and with a good postoperative course. In moyamoya syndrome associated with Williams syndrome, adequate perioperative management of both the moyamoya arteries and the cardiovascular abnormalities is important to prevent complications. CONCLUSION: This was the first report on a case in which moyamoya syndrome associated with Williams syndrome was confirmed by the presence of a heterozygous RNF213 mutation. Similar to the workup of moyamoya disease, confirmation of RNF213 mutation in Williams syndrome may be useful in predicting the development of moyamoya syndrome that can lead to severe complications.

A Triple High Throughput Screening for Extracellular Vesicle Inducing Agents With Immunostimulatory Activity.

Extracellular vesicles (EVs) play an important role in intercellular communication and regulation of cells, especially in the immune system where EVs can participate in antigen presentation and may have adjuvant effects. We aimed to identify small molecule compounds that can increase EV release and thereby enhance the immunogenicity of vaccines. We utilized a THP-1 reporter cell line engineered to release EV-associated tetraspanin (CD63)-Turbo-luciferase to quantitatively measure EVs released in culture supernatants as a readout of a high throughput screen (HTS) of 27,895 compounds. In parallel, the cytotoxicity of the compounds was evaluated by PrestoBlue dye assay. For screening immunostimulatory potency, we performed two additional independent HTS on the same compound library using NF-κB and interferon-stimulated response element THP-1 reporter cell lines. Hit compounds were then identified in each of the 3 HTS's, using a "Top X″ and a Gaussian Mixture Model approach to rule out false positive compounds and to increase the sensitivity of the hit selection. Thus, 644 compounds were selected as hits which were further evaluated for induction of IL-12 in murine bone-marrow derived dendritic cells (mBMDCs) and for effects of cell viability. The resulting 130 hits were then assessed from a medicinal chemistry perspective to remove compounds with functional group liabilities. Finally, 80 compounds were evaluated as vaccine adjuvants in vivo using ovalbumin as a model antigen. We analyzed 18 compounds with adjuvant activity for their ability to induce the expression of co-stimulatory molecules on mBMDCs. The full complement of data was then used to cluster the compounds into 4 distinct biological activity profiles. These compounds were also evaluated for quantitation of EV release and spider plot overlays were generated to compare the activity profiles of compounds within each cluster. This tiered screening process identified two compounds that belong to the 4-thieno-2-thiopyrimidine scaffold with identical screening profiles supporting data reproducibility and validating the overall screening process. Correlation patterns in the adjuvanticity data suggested a role for CD63 and NF-κB pathways in potentiating antigen-specific antibody production. Thus, our three independent cell-based HTS campaigns led to identification of immunostimulatory compounds that release EVs and have adjuvant activity.

Glyco-Nanoadjuvants: Impact of Linker Length for Conjugating a Synthetic Small-Molecule TLR7 Ligand to Glyco-Nanoparticles on Immunostimulatory Effects.

Immunotherapy has become a powerful clinical strategy for treating infectious diseases and cancer. Synthetic small-molecule toll-like receptor 7 (TLR7) ligands are attractive candidates as immunostimulatory agents for immunotherapy. TLR7 is mainly localized in intracellular endosomal compartments so that the formulation of their small-molecule ligands with macromolecules enhances endocytic uptake of TLR7 ligands and improves the pharmaceutical properties. Previously, we demonstrated that gold nanoparticles co-immobilized with a TLR7 ligand derivative, that is, a conjugate of synthetic small-molecule TLR7 ligand (1V209) and thioctic acid (TA) via 4,7,10-trioxa-1,13-tridecanediamine, and α-mannose (1V209-αMan-GNPs: glyco-nanoadjuvants) significantly enhances immunostimulatory effects. In the present study, we designed a second-generation glyco-nanoadjuvant that possesses a poly(ethylene glycol) (PEG) chain as a spacer between 1V209 and GNPs and investigated the impact of linker length in 1V209 derivatives on the immunostimulatory activities. We used different chain lengths of PEG (n = 3, 5, 11, or 23) as spacers between 1V209 and thioctic acid to prepare four 1V209-αMan-GNPs. In the in vitro study using primary mouse bone-marrow-derived dendritic cells, 1V209-αMan-GNPs that immobilized with longer 1V209 derivatives, especially the 1V209 derivative possessing PEG23 (1V209-PEG23-TA), showed the highest potency toward induction both for interleukin-6 and type I interferon production than those derivatives with shorter PEG chains. Furthermore, 1V209-αMan-GNPs that immobilized with 1V209-PEG23-TA showed significantly higher adjuvant effects for inducing both humoral and cell-mediated immune responses against ovalbumin in the in vivo immunization study. These results indicate that the linker length for immobilizing small-molecule TLR7 ligand on the GNPs significantly affects the adjuvant activity of 1V209-αMan-GNPs and that 1V209-αMan-GNPs immobilized with 1V209-PEG-23-TA could be superior adjuvants for immunotherapies.

Small Molecule Calcium Channel Activator Potentiates Adjuvant Activity.

There remains an unmet need for reliable fully synthetic adjuvants that increase lasting protective immune responses from vaccines. We previously reported a high-throughput screening for small molecules that extended nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) activation after a Toll-like receptor 4 (TLR4) ligand, lipopolysaccharide (LPS), stimulation using a human myeloid reporter cell line. We identified compounds with a conserved aminothiazole scaffold including 2D216 [N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide], which increased murine antigen-specific antibody responses when used as a co-adjuvant with LPS. Here, we examined the mechanism of action in human cells. Although 2D216 activated the major mitogen-activated protein kinases, it did not interact with common kinases and phosphatases and did not stimulate many of the pattern recognition receptors (PRRs). Instead, the mechanism of action was linked to intracellular Ca2+ elevation via Ca2+ channel(s) at the plasma membrane and nuclear translocation of the nuclear factor of activated T-cells (NFAT) as supported by RNA-seq data, analysis by reporter cells, Ca2+ flux assays, and immunoblots. Interestingly, 2D216 had minimal, if any, activity on Jurkat T cells but induced cytokine production and surface expression of costimulatory molecules on cells with antigen-presenting functions. A small series of analogs of 2D216 were tested for the ability to enhance a TLR4 ligand-stimulated autologous mixed lymphocyte reaction (MLR). In the MLR, 2E151, N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-((4-propylpiperidin-1-yl)sulfonyl)benzamide, was more potent than 2D216. These results indicate that a small molecule that is not a direct PRR agonist can act as a co-adjuvant to an approved adjuvant to enhance human immune responses via a complementary mechanism of action.

Neuroendoscopic fenestration for intracranial unilocular cysts and isolated lateral ventricles: four pediatric cases.

The purpose of treatment for unilocular intracranial cysts (UICs) is to release elevated intracranial pressure. Neuroendoscopic fenestration (NF) is one of the most effective and minimally invasive options for treating UICs, especially in young children; however, the optimal location and number of fenestrations, the necessity of using endoscopic third ventriculostomy (ETV) in combination with fenestration, and the course of treatment are not well known. We retrospectively reviewed the hospital records between 2012 and 2019. The patients were studied in terms of sex, age at surgery, preoperative symptoms, cyst localization and size, course of treatment, ventricular diameter, developmental assessment, anatomical location, and the number of fenestrations. There were four eligible patients in the relevant period: two boys and two girls. The median age at the time of surgery was 16 months. With regard to the location of the cysts, there were two cases of cavum velum interpositum (CVI), one case of quadrigeminal cistern, and one case of an isolated lateral ventricle. The most common preoperative finding was an enlarged head circumference. All the patients were treated with NF, including one case of reoperation after open head surgery. Postoperatively, we used the frontal and occipital horn ratio (FOHR) to evaluate the ventricular size. The average reduction in the FOHR was 0.003. In the most recent developmental assessment or examination during the follow-up period, two patients showed normal development, and two patients showed developmental delay. Based on our past experience and reports, we believe that it is recommended to perform two fenestrations for a single cyst. This is because it creates a flow of cerebrospinal fluid (CSF) within the cyst into normal CSF reflux. For lesions with obstruction of the aqueduct, such as cysts in the quadrigeminal cistern, ETV should be considered if it can be performed safely, in preparation for the worsening of hydrocephalus due to obstruction by enlargement of the cyst.

Hemodynamic Change due to Vessel Straightening Immediately after LVIS Jr. Deployment for an Anterior Communicating Artery Aneurysm.

Objective: Stent-assisted coil embolization for cerebral aneurysms may lead to straightening of the parent vessel. However, detailed reports documenting the hemodynamic change in bifurcation type aneurysms due to straightening of the parent vessel immediately after stent deployment are scarce. Case Presentation: A 48-year-old woman with a history of polycystic kidney disease underwent aneurysm neck clipping with left frontotemporal craniotomy for a ruptured bifurcation-type anterior communicating artery (AComA) aneurysm. Angiography 18 days after clipping showed a recurrent AComA aneurysm, for which stent-assisted coil embolization was performed. Straightening of the parent vessel immediately after deployment of a low-profile visualized intraluminal support junior (LVIS Jr.) stent from the AComA to the A1 segment of the right anterior cerebral artery was confirmed by working projection angiography. The aneurysm was easily embolized with coils with the support of the stent covering the aneurysm neck. The embolization was finished with a slight dome filling of the aneurysm. The parent vessel angle in 3D angiography changed from 90° before stent deployment to 160° immediately after stent deployment. Angiography 2 months after embolization showed the aneurysm with a complete occlusion and the parent vessel angle of 170° in a 3D image. Conclusion: The hemodynamic change in a bifurcation-type AComA aneurysm due to straightening of the parent vessel immediately after the LVIS Jr. stent deployment led to the covering of the aneurysm neck, resulting in good coil embolization, to which the vessel mobility and the stenting method may have contributed.

Gradual dilatation of an occluded transverse sinus associated with dural arteriovenous fistula after balloon angioplasty with sinus packing: A case report.

BACKGROUND: There is no consensus as to whether balloon angioplasty alone or stent placement is effective for sinus occlusion associated with dural arteriovenous fistula (DAVF). Herein, we first report a case of transverse sinus occlusion associated with DAVF in which gradual sinus dilatation was observed after balloon angioplasty with embolization of the affected sinus with shunt flow. CASE PRESENTATION: A 69-year-old man presented with executive dysfunction. Magnetic resonance imaging revealed left transverse sinus-sigmoid sinus DAVF with occlusion of the left jugular vein and right transverse sinus. Before endovascular treatment, the patient had symptomatic epilepsy and subarachnoid hemorrhage. Retrograde leptomeningeal venous drainage disappeared with packing of the left transverse sinus-sigmoid sinus. Subsequently, balloon angioplasty of the right occluded transverse sinus was performed to maintain the normal venous drainage and remaining shunt outflow. Dilatation of the right transverse sinus was poor immediately after surgery. However, angiography after 10 days and 6 months revealed gradual dilatation of the right transverse sinus. CONCLUSION: Sinus occlusion, which is thought to be caused by sinus hypertension associated with DAVF rather than chronic organized thrombosis or thrombophilia, may dilate over time after balloon angioplasty and shunt flow reduction if occluded sinus is necessary for facilitating normal venous drainage.

Low-profile visualized intraluminal support Blue stenting within a Neuroform Atlas stent for a large wide-necked aneurysm: A case report and a bench-top experiment.

Low-profile visualized intraluminal support deployment in an Enterprise has been reported; however, that in an Atlas has yet to be in detail. Enterprise has a closed-cell design, while Atlas has an open-cell design. We detail here a case of a large wide-necked aneurysm treated by coil embolization with low-profile visualized intraluminal support Blue deployment within a Neuroform Atlas and a bench-top experiment using a silicon tube to test low-profile visualized intraluminal support, Atlas, Enterprise, and their combinations. A better low-profile visualized intraluminal support expansion was achieved by simultaneously pushing the wire and the system within the Atlas placed at the aneurysm neck, which resulted in an increased metal coverage of the aneurysm neck and a shorter transition zone with low metal coverage at both ends of the aneurysm neck. This technique may enable a high metal coverage by low-profile visualized intraluminal support expansion without restriction by the Atlas and contribute to aneurysm occlusion by increasing the flow-diverting effect.

Small Molecule Potentiator of Adjuvant Activity Enhancing Survival to Influenza Viral Challenge.

As viruses continue to mutate the need for rapid high titer neutralizing antibody responses has been highlighted. To meet these emerging threats, agents that enhance vaccine adjuvant activity are needed that are safe with minimal local or systemic side effects. To respond to this demand, we sought small molecules that would sustain and improve the protective effect of a currently approved adjuvant, monophosphoryl lipid A (MPLA), a Toll-like receptor 4 (TLR4) agonist. A lead molecule from a high-throughput screen, (N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide, was identified as a hit compound that sustained NF-κB activation by a TLR4 ligand, lipopolysaccharide (LPS), after an extended incubation (16 h). In vitro, the resynthesized compound (2D216) enhanced TLR4 ligand-induced innate immune activation and antigen presenting function in primary murine bone marrow-derived dendritic cells without direct activation of T cells. In vivo murine vaccination studies demonstrated that compound 2D216 acted as a potent co-adjuvant when used in combination with MPLA that enhanced antigen-specific IgG equivalent to that of AS01B. The combination adjuvant MPLA/2D216 produced Th1 dominant immune responses and importantly protected mice from lethal influenza virus challenge. 2D216 alone or 2D216/MPLA demonstrated minimal local reactogenicity and no systemic inflammatory response. In summary, 2D216 augmented the beneficial protective immune responses of MPLA as a co-adjuvant and showed an excellent safety profile.

Structure-activity relationship studies in substituted sulfamoyl benzamidothiazoles that prolong NF-κB activation.

In the face of emerging infectious diseases, there remains an unmet need for vaccine development where adjuvants that enhance immune responses to pathogenic antigens are highly desired. Using high-throughput screens with a cell-based nuclear factor κB (NF-κB) reporter assay, we identified a sulfamoyl benzamidothiazole bearing compound 1 that demonstrated a sustained activation of NF-κB after a primary stimulus with a Toll-like receptor (TLR)-4 agonist, lipopolysaccharide (LPS). Here, we explore systematic structure-activity relationship (SAR) studies on compound 1 that indicated the sites on the scaffold that tolerated modification and yielded more potent compounds compared to 1. The selected analogs enhanced release of immunostimulatory cytokines in the human monocytic cell line THP-1 cells and murine primary dendritic cells. In murine vaccination studies, select compounds were used as co-adjuvants in combination with the Food and Drug Administration approved TLR-4 agonistic adjuvant, monophosphoryl lipid A (MPLA) that showed significant enhancement in antigen-specific antibody titers compared to MPLA alone. Additionally, our SAR studies led to identification of a photoaffinity probe which will aid the target identification and mechanism of action studies in the future.

Mitochondria-dependent synthetic small-molecule vaccine adjuvants for influenza virus infection.

Vaccine adjuvants enhance and prolong pathogen-specific protective immune responses. Recent reports indicate that host factors-such as aging, pregnancy, and genetic polymorphisms-influence efficacies of vaccines adjuvanted with Toll-like receptor (TLR) or known pattern-recognition receptor (PRR) agonists. Although PRR independent adjuvants (e.g., oil-in-water emulsion and saponin) are emerging, these adjuvants induce some local and systemic reactogenicity. Hence, new TLR and PRR-independent adjuvants that provide greater potency alone or in combination without compromising safety are highly desired. Previous cell-based high-throughput screenings yielded a small molecule 81 [N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide], which enhanced lipopolysaccharide-induced NF-κB and type I interferon signaling in reporter assays. Here compound 81 activated innate immunity in primary human peripheral blood mononuclear cells and murine bone marrow-derived dendritic cells (BMDCs). The innate immune activation by 81 was independent of TLRs and other PRRs and was significantly reduced in mitochondrial antiviral-signaling protein (MAVS)-deficient BMDCs. Compound 81 activities were mediated by mitochondrial dysfunction as mitophagy inducers and a mitochondria specific antioxidant significantly inhibited cytokine induction by 81. Both compound 81 and a derivative obtained via structure-activity relationship studies, 2F52 [N-benzyl-N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] modestly increased mitochondrial reactive oxygen species and induced the aggregation of MAVS. Neither 81 nor 2F52 injected as adjuvants caused local or systemic toxicity in mice at effective concentrations for vaccination. Furthermore, vaccination with inactivated influenza virus adjuvanted with 2F52 demonstrated protective effects in a murine lethal virus challenge study. As an unconventional and safe adjuvant that does not require known PRRs, compound 2F52 could be a useful addition to vaccines.

Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release.

Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After four passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z' factor (average of 2-day screen, 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.

Oral administration of PEGylated TLR7 ligand ameliorates alcohol-associated liver disease via the induction of IL-22.

Effective therapies for alcohol-associated liver disease (ALD) are limited; therefore, the discovery of new therapeutic agents is greatly warranted. Toll-like receptor 7 (TLR7) is a pattern recognition receptor for single-stranded RNA, and its activation prevents liver fibrosis. We examined liver and intestinal damage in Tlr7-/- mice to determine the role of TLR7 in ALD pathogenesis. In an alcoholic hepatitis (AH) mouse model, hepatic steatosis, injury, and inflammation were induced by chronic binge ethanol feeding in mice, and Tlr7 deficiency exacerbated these effects. Because these results demonstrated that endogenous TLR7 signaling activation is protective in the AH mouse model, we hypothesized that TLR7 activation may be an effective therapeutic strategy for ALD. Therefore, we investigated the therapeutic effect of TLR7 agonistic agent, 1Z1, in the AH mouse model. Oral administration of 1Z1 was well tolerated and prevented intestinal barrier disruption and bacterial translocation, which thus suppressed ethanol-induced hepatic injury, steatosis, and inflammation. Furthermore, 1Z1 treatment up-regulated the expression of antimicrobial peptides, Reg3b and Reg3g, in the intestinal epithelium, which modulated the microbiome by decreasing and increasing the amount of Bacteroides and Lactobacillus, respectively. Additionally, 1Z1 up-regulated intestinal interleukin (IL)-22 expression. IL-22 deficiency abolished the protective effects of 1Z1 in ethanol-induced liver and intestinal damage, suggesting intestinal IL-22 as a crucial mediator for 1Z1-mediated protection in the AH mouse model. Collectively, our results indicate that TLR7 signaling exerts protective effects in the AH mouse model and that a TLR7 ligand, 1Z1, holds therapeutic potential for the treatment of AH.

Glyco-Nanoadjuvants: Sugar Structures on Carriers of a Small Molecule TLR7 Ligand Affect Their Immunostimulatory Activities.

Toll-like receptors (TLRs) are pattern recognition receptors that activate innate immunity, and their ligands are promising adjuvants for vaccines and immunotherapies. Small molecule TLR7 ligands are ideal vaccine adjuvants as they induce not only proinflammatory cytokines but also type I interferons. However, their application has only been approved for local administration due to severe systemic immune-related adverse events. In a previous study, we prepared the gold nanoparticles coimmobilized with synthetic small molecule TLR7 ligand, 1V209, and α-mannose (1V209-αMan-GNPs). 1V209-αMan-GNPs were selectively delivered via a cell surface sugar-binding protein, mannose receptor, which enabled selective delivery of TLR7 ligands to immune cells. Besides the mannose receptor, immune cells express various sugar-binding proteins such as macrophage galactose binding lectins and sialic acid-binding immunoglobulin-type lectins and recognize distinct sugar structures. Hence, in the present study, we investigated whether sugar structures on GNPs affect the efficiency and selectivity of intracellular delivery and subsequent immunostimulatory potencies. Five neutral sugars and two sialosides were selected and each sugar was coimmobilized with 1V209 onto GNPs (1V209-SGNPs) and their innate immunostimulatory potencies were compared to that of 1V209-αMan-GNPs. The in vitro study using mouse bone marrow derived dendritic cells (BMDCs) demonstrated that α-glucose, α-N-acetylglucosamine, or α-fucose immobilized 1V209-SGNPs increased interleukin-6 and type I interferon release similar to that of 1V209-αMan-GNPs, whereas galacto-type sugar immobilized 1V209-SGNPs predominantly enhanced type I interferon release. In contrast, sialoside immobilized 1V209-SGNPs did not enhance the potency of 1V209. In the in vivo immunization study using ovalbumin as a model antigen, neutral sugar immobilized 1V209-SGNPs induced comparable T helper-1 immune response to that of 1V209-αMan-GNPs and by 10-fold higher than that of sialoside immobilized 1V209-SGNPs. These results indicate that the sugar structures on 1V209-SGNPs affect their immunostimulatory activities, and functionalization of the carrier particles is important to shape immune responses.