The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus.

Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5'-ε region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5'-ε region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this ε region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.

Lymphatic Endothelial Cells Produce Chemokines in Response to the Lipid Nanoparticles Used in RNA Vaccines.

RNA vaccines based on Lipid nanoparticles (LNP) were put into practical use within only one year after the global outbreak of the coronavirus disease 2019 (COVID-19). This success of RNA vaccine highlights the utility of an mRNA delivery system as a vaccination strategy. Potent immunostimulatory activity of LNPs (i.e., inflammation occurring at the injection site and the production of inflammatory cytokines) have recently been reported. However, we have only limited knowledge concerning which cells are responsible for responding to the LNPs. We report herein on in vitro chemokine production from non-immune cells in response to exposure to LNPs. In this study, SM-102, an ionizable lipid that is used in the approved RNA vaccine for the clinical usage of COVID-19 mRNA vaccine, was used. Immortalized mouse lymphatic endothelial cells (mLECs) or professional antigen presenting cells (APCs) such as RAW 264.7 monocyte/macrophage cells were incubated with LNPs that contained no mRNA. As a result, chemokines involved in the recruitment of monocytes/neutrophils were produced only by the mLECs following the LNP treatment. These findings indicate that LEC appear to serve as the cell that sends out initial signals to response LNPs.

Reactivation of Anticancer Immunity by Resetting Interorgan Crosstalk in Immune-Suppressive Cells with a Nanoparticulated Anti-Inflammatory Drug.

The reactivation of anticancer immunity is a fundamental principle in cancer immunotherapy as evidenced by the use of immune checkpoint inhibitors (ICIs). While treatment with the ICIs is shown to have remarkable and durable therapeutic effects in the responders, the low objective response rate (<40%) continues to be a major problem. Since myeloid-derived suppressor cells (MDSCs), heterogenous cells with strong immunosuppressive activity that originate in the hematopoietic system, suppress the anticancer immunity via parallel immune checkpoint-dependent and independent pathways, these cells are potential targets for improving the efficacy of cancer immunotherapy. In this study, it is demonstrated that MDSCs can be depleted by delivering synthetic glucocorticoid dexamethasone to phagocytic cells in the spleen using a lipid nanoparticle. Since the interaction of nanoparticles with T cells is intrinsically poor, this strategy also enables the "detargeting" from T cells, thus avoiding the nonspecific suppression of cytotoxic immune responses against cancer cells. In addition to the direct anticancer effect of the nanoparticulated dexamethasone, their synergistic anticancer effect with ICIs is also reported.

A Lipid Nanoparticle-Based Method for the Generation of Liver-Specific Knockout Mice.

Knockout mice are useful tools that can provide information about the normal function of genes, including their biochemical, developmental, and physiological roles. One problem associated with the generation of knockout mice is that the loss of some genes of interest produces a lethal phenotype. Therefore, the use of conditioned knockout mice, in which genes are disrupted in specific organs, is essential for the elucidation of disease pathogenesis and the verification of drug targets. In general, conditional knockout mice are produced using the Cre/loxP system; however, the production of the large numbers of Cre/flox knockout and control mice required for analysis requires substantial time and effort. Here, we describe the generation of liver-specific conditional knockout mice via the introduction of lipid nanoparticles encapsulating Cre mRNA into the liver of floxed mice. This technique does not require the production of offspring by mating floxed mice and is therefore more convenient than the conventional method. The results presented here demonstrate that the LNP-based method enables liver-specific gene knockout in a short period of time.

Human Immortalized Cell-Based Blood-Brain Barrier Spheroid Models Offer an Evaluation Tool for the Brain Penetration Properties of Macromolecules.

Blood-brain barrier (BBB)-permeable middle- or macromolecules (middle/macromolecules) have recently attracted significant attention as new drug delivery carriers into the human brain via receptor-mediated transcytosis (RMT). During the development process of such carriers, it is necessary to thoroughly evaluate their human BBB permeability levels. In such evaluations, our recently established human immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models) have shown high potential. However, the specifics of those capabilities have yet to be elucidated. Therefore, in this study, we characterize the ability of the hiMCS-BBB models to evaluate RMT-mediated BBB penetration properties of middle/macromolecules. More specifically, we began by validating transferrin receptor (TfR)-mediated RMT functionalities using transferrin in the hiMCS-BBB models and then examined the BBB permeability levels of MEM189 antibodies (known BBB-permeable anti-TfR antibodies). The obtained results showed that, as with the case of transferrin, temperature-dependent uptake of MEM189 antibodies was observed in the hiMCS-BBB models, and the extent of that uptake increased in a time-dependent manner until reaching a plateau after around 2 h. To further expand the evaluation applicability of the models, we also examined the BBB permeability levels of the recently developed SLS cyclic peptide and observed that peptide uptake was also temperature-dependent. To summarize, our results show that the hiMCS-BBB models possess the ability to evaluate the RMT-mediated BBB-permeable properties of antibodies and peptides and thus have the potential to provide valuable tools for use in the exploration and identification of middle/macromolecules showing excellent BBB permeability levels, thereby contributing powerfully to the development of new drug delivery carriers for transporting drugs into the human brain.

Development of Sentinel LN Imaging with a Combination of HAase Based on a Comprehensive Analysis of the Intra-lymphatic Kinetics of LPs.

The sentinel lymph node (LN) is the first LN to which lymph fluid flows from tumor tissue. We identified the key parameters of liposomes (LPs) that affect their accumulation in regional (primary) LNs with minimum leakage to its connecting (secondary) LNs by a comprehensive analysis of the LN-to-LN trafficking of LPs with various surface charges and various sizes. We used a lymphatic flow-modified (LFM) mouse that allows for the chronological analysis of inguinal (primary) LN-to-axillary (secondary) LN at the body surface. As a result, the anionic medium-sized LPs (130 nm on average) exhibited the highest accumulation in the primary LNs. A mechanism-based analysis revealed that CD169-positive macrophages in LNs were the dominant cell population that captures anionic LPs. Sentinel LN imaging was also performed by the intratumoral injection of fluorescent medium-sized anionic LPs using a breast cancer orthotopic model. In comparison with the typically used contrast agent indocyanine green, the anionic LPs were detected in sentinel LNs with a high sensitivity. Additionally, the co-injection of hyaluronidase significantly improved the sensitivity of detection of the fluorescent LPs in sentinel LNs. In conclusion, medium-sized anionic LPs combined with hyaluronidase represents a potent strategy for investigating sentinel LNs.

Cellular Binding and Internalization Assay for an Anti-FcγRIIB Antibody Using Human Liver Non-parenchymal Cells.

A cellular assay for evaluating the binding and internalization of biologics using primary human liver sinusoidal endothelial cells (LSEC) is not readily available, since human LSEC generally lose their receptor expression and internalization activity during the purifying processes and cell culturing. Here, we propose a novel cell-based assay using human liver non-parenchymal cells (NPC) as an alternative method using LSEC. To identify the LSEC population, NPC were stained with CD31 and CD45, and analyzed by flow cytometry. The expression of Fc gamma receptor IIB (FcγRIIB), one of the LSEC markers was detected in the CD31-positive and the CD45-negative fractions. The concentration-dependent binding and internalization of the anti-FcγRIIB antibody was also quantified in the LSEC fraction in human NPC. Saturated binding and internalization curves were obtained for the anti-FcγRIIB antibody. In the case of the negative control antibody, however, binding and internalization were negligible. The findings reported here indicate that cell-based assays using fresh human liver NPC will be useful for evaluating the binding and internalization of biologics as well as for determining pharmacokinetic parameters.

Lipid nanoparticles-encapsulated brain-derived neurotrophic factor mRNA delivered through the round window niche in the cochleae of guinea pigs.

The treatment of sensorineural hearing loss (SNHL) may be achieved via the application of a cochlear implant (CI) that allows the electrical stimulation of spiral ganglion neurons (SGNs). Nevertheless, the efficacy of CIs is limited by the degeneration of SGNs following SNHL. Although the application of exogenous neurotrophic factors has been reported to decrease SGN degeneration, non-invasive targeted drug delivery systems are required to achieve effective results. In this study, an SS-cleavable proton-activated lipid-like material [ssPalm; a neutral lipid nanoparticle (LNP)], was loaded with mRNA, and the efficacy of this material as a delivery system was investigated. Our results showed that LNPssPalm carrying brain-derived neurotrophic factor (BDNF) mRNA was suitable for the treatment of inner ear diseases, preventing the degeneration of SGNs. In conclusion, this modern nanotechnology-based bioconjugation system, LNPssPalm, is a potential non-invasive targeted therapy allowing the delivering biomaterials to specific structures within the inner ear for the treatment of SHNL.

Proteomics Analysis of Lymphatic Metastasis-Related Proteins Using Highly Metastatic Human Melanoma Cells Originated by Sequential in Vivo Implantation.

Metastasis of cancer cells to lymph nodes (LN) is a common modality of metastasis in clinical settings, but the mechanisms involved in lymphatic metastasis remain unclear compared to hematogenous metastasis to bones and the brain. To elucidate the molecular mechanisms responsible for melanoma LN metastasis, we first generated LN metastasis-prone melanoma cells (C8161F2) by the sequential in vivo transplantation of parental melanoma cells (C8161F0). Although the in vitro/in vivo proliferative potential of these melanoma cells were similar, the metastatic potential of the C8161F2 for LNs was significantly enhanced. We then conducted a proteomics analysis to identify the proteins and pathways that contribute to LN metastasis. We identified six proteins (three: up-regulated and three: down-regulated) whose expressions were statistically significantly different by more than 2-fold in the two cell groups. Some of these genes are responsible for the activation of the transforming growth factor-ß (TGF-ß)-related pathway, a well-known inducer of epithelial-mesenchymal transition (EMT). In addition, a gene ontology analysis revealed that the enhanced cell-cell adhesion appears to be involved in lymphatic metastasis. In conclusion, we established highly lymphatic metastatic melanoma cells, which would be valuable for studies of the molecular mechanisms responsible for lymphatic metastasis.

Development, Characterization and Potential Applications of a Multicellular Spheroidal Human Blood-Brain Barrier Model Integrating Three Conditionally Immortalized Cell Lines.

In vitro blood-brain barrier (BBB) models are essential research tools for use in developing brain-targeted drugs and understanding the physiological and pathophysiological functions of the BBB. To develop BBB models with better functionalities, three-dimensional (3D) culture methods have gained significant attention as a promising approach. In this study, we report on the development of a human conditionally immortalized cell-based multicellular spheroidal BBB (hiMCS-BBB) model. After being seeded into non-attachment culture wells, HASTR/ci35 (astrocytes) and HBPC/ci37 cells (brain pericytes) self-assemble to form a spheroid core that is then covered with an outer monolayer of HBMEC/ci18 cells (brain microvascular endothelial cells). The results of immunocytochemistry showed the protein expression of several cellular junction and BBB-enriched transporter genes in HBMEC/ci18 cells of the spheroid model. The permeability assays showed that the hiMCS-BBB model exhibited barrier functions against the penetration of dextran (5 and 70 kDa) and rhodamine123 (a P-glycoprotein substrate) into the core. On the other hand, facilitation of 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG; a fluorescent glucose analog) uptake was observed in the hiMCS-BBB model. Furthermore, tumor necrosis factor-alpha treatment elicited an inflammatory response in HBMEC/ci18 cells, thereby suggesting that BBB inflammation can be recapitulated in the hiMCS-BBB model. To summarize, we have developed an hiMCS-BBB model that possesses fundamental BBB properties, which can be expected to provide a useful and highly accessible experimental platform for accelerating various BBB studies.

In vivo evaluation of intestinal human CYP3A inhibition by macrolide antibiotics in CYP3A-humanised mice.

It is important to predict drug-drug interactions via inhibition of intestinal cytochrome P450 3A (CYP3A) which is a determinant of bioavailability of orally administered CYP3A substrates. However, inhibitory effects of macrolide antibiotics on CYP3A-mediated metabolism are not entirely identical between humans and rodents.We investigated the effects of macrolide antibiotics, clarithromycin and erythromycin, on in vitro and in vivo metabolism of triazolam, a CYP3A substrate, in CYP3A-humanised mice generated by using a mouse artificial chromosome vector carrying a human CYP3A gene.Metabolic activities of triazolam were inhibited by macrolide antibiotics in liver and intestine microsomes of CYP3A-humanised mice.The area under the plasma concentration-time curve ratios of 4-hydroxytriazolam to triazolam after oral dosing of triazolam were significantly decreased by multiple administration of macrolide antibiotics. The plasma concentrations ratios of α-hydroxytriazolam and 4-hydroxytriazolam to triazolam in portal blood were significantly decreased by multiple administration of clarithromycin in CYP3A-humanised mice.These results suggest that intestinal CYP3A activity was inhibited by macrolide antibiotics in CYP3A-humanised mice in vitro and in vivo. The plasma concentrations of triazolam and its metabolites in the portal blood of CYP3A-humanised mice would be useful for direct evaluation of intestinal CYP3A-mediated drug-drug interactions.

Effects on Metabolism in Astrocytes Caused by cGAMP, Which Imitates the Initial Stage of Brain Metastasis.

The second messenger 2'3'-cyclic-GMP-AMP (cGAMP) is thought to be transmitted from brain carcinomas to astrocytes via gap junctions, which functions to promote metastasis in the brain parenchyma. In the current study, we established a method to introduce cGAMP into astrocytes, which simulates the state of astrocytes that have been invaded by cGAMP around tumors. Astrocytes incorporating cGAMP were analyzed by metabolomics, which demonstrated that cGAMP increased glutamate production and astrocyte secretion. The same trend was observed for γ-aminobutyric acid (GABA). Conversely, glutamine production and secretion were decreased by cGAMP treatment. Due to the fundamental role of astrocytes in regulation of the glutamine-glutamate cycle, such metabolic changes may represent a potential mechanism and therapeutic target for alteration of the central nervous system (CNS) environment and the malignant transformation of brain carcinomas.

Clinicopathologic and Immunohistochemical Findings from Autopsy of Patient with COVID-19, Japan.

An autopsy of a patient in Japan with coronavirus disease indicated pneumonia lung pathology, manifested as diffuse alveolar damage. We detected severe acute respiratory syndrome coronavirus 2 antigen in alveolar epithelial cells and macrophages. Coronavirus disease is essentially a lower respiratory tract disease characterized by direct viral injury of alveolar epithelial cells.

Vitamin E Scaffolds of pH-Responsive Lipid Nanoparticles as DNA Vaccines in Cancer and Protozoan Infection.

DNA vaccinations are promising strategies for treating diseases that require cellular immunity (i.e., cancer and protozoan infection). Here, we report on the use of a liposomal nanocarrier (lipid nanoparticles (LNPs)) composed of an SS-cleavable and pH-activated lipidlike material (ssPalm) as an in vivo DNA vaccine. After subcutaneous administration, the LNPs containing an ssPalmE, an ssPalm with vitamin E scaffolds, elicited a higher gene expression activity in comparison with the other LNPs composed of the ssPalms with different hydrophobic scaffolds. Immunization with the ssPalmE-LNPs encapsulating plasmid DNA that encodes ovalbumin (OVA, a model tumor antigen) or profilin (TgPF, a potent antigen of Toxoplasma gondii) induced substantial antitumor or antiprotozoan effects, respectively. Flow cytometry analysis of the cells that had taken up the LNPs in draining lymph nodes (dLNs) showed that the ssPalmE-LNPs were largely taken up by macrophages and a small number of dendritic cells. We found that the transient deletion of CD169+ macrophages, a subpopulation of macrophages that play a key role in cancer immunity, unexpectedly enhanced the activity of the DNA vaccine. These data suggest that the ssPalmE-LNPs are effective DNA vaccine carriers, and a strategy for avoiding their being trapped by CD169+ macrophages will be a promising approach for developing next-generation DNA vaccines.

Development of an SS-Cleavable pH-Activated Lipid-Like Material (ssPalm) as a Nucleic Acid Delivery Device.

Gene and nucleic acid-based medication is an ultimate strategy in the field of personalized medicine. A gene or short interference RNA (siRNA) molecule needs to be delivered to the appropriate organelle (i.e., nucleus and cytoplasm, respectively). We recently focused on improving the intrinsic activity of my original material (ssPalm) in terms of endosomal/lysosomal membrane destabilization activity by chemically modifying the tertiary amine structure. In parallel, I have been expanding the range of applications of ssPalms. The first application is a DNA or RNA vaccine. My crucial finding is that the vitamin E-scaffold ssPalm (ssPalmE) is highly immune-stimulative when combined with DNA. Thereafter, I redesigned the hydrophobic scaffold structure, and found that an oleic acid-scaffold ssPalm (ssPalmO) can confer anti-inflammatory characteristics. Based on this result, I further upgraded the ssPalmO, by inserting a newly designed linker with self-degradable properties.

[Two Cases of Advanced Gastric Cancer Diagnosed as Pathological Complete Response after Preoperative Chemotherapy with S-1 and Oxaliplatin].

We herein report 2 cases of gastric cancer treated by S-1 and oxaliplatin combination therapy before later undergoing gastrectomy. The pathological results of both cases demonstrated complete response. Case 1 had a giant tumor which was suspected to have invaded the pancreas. Case 2 was associated with extensive lymph node metastasis. Based on the findings of these 2 cases, preoperative chemotherapy with S-1 and oxaliplatin for advanced gastric cancer shows sufficient efficacy.

[Case of Peritoneal Dissemination from Gastrointestinal Stromal Tumor of Jejunum Treated with Repeated Non-Curative Surgery].

A 71-year-old male presented with abdominal distension and fever to our hospital. Abdominal CT revealed a huge tumor in abdomen, and non-curative surgery was performed. Peritoneal dissemination was widespread and the tumor invaded the bladder and sigmoid-colon mesenterium. Two months after the initial surgery, CT showed liver metastasis, and oral administration of imatinib mesylate was started. The peritoneal dissemination and liver metastasis showed a decrease, and this was well controlled for 45 months without severe side effects. Abdominal CT revealed peritoneal dissemination in the ileocecum after 43 months since the administration of imatinib. Therefore, sunitinib treatment was initiated. After 3 months of sunitinib administration, the tumor perforated. Emergency operation was performed to resect the ileocecum, and sunitinib was continued for 1 year. In GIST with liver metastasis and peritoneal dissemination, repeated surgical resection combined with chemotherapy is important to improve the patient's survival.

A Human Immortalized Cell-Based Blood-Brain Barrier Triculture Model: Development and Characterization as a Promising Tool for Drug-Brain Permeability Studies.

Brain microvascular endothelial cells (BMEC), together with astrocytes and pericytes, form the blood-brain barrier (BBB) that strictly restricts drug penetration into the brain. Therefore, in central nervous system drug development, the establishment of an in vitro human BBB model for use in studies estimating the in vivo human BBB permeability of drug candidates has long been awaited. The current study developed and characterized a human immortalized cell-based BBB triculture model, termed the "hiBBB" model. To set up the hiBBB model, human immortalized BMEC (HBMEC/ci18) were cocultured with human immortalized astrocytes (HASTR/ci35) and brain pericytes (HBPC/ci37) in a transwell system. The trans-endothelial electrical resistance of the hiBBB model was 134.4 ± 5.5 (Ω × cm2), and the efflux ratios of rhodamine123 and dantrolene were 1.72 ± 0.11 and 1.72 ± 0.45, respectively, suggesting that the hiBBB model possesses essential cellular junction and efflux transporter functions. In BBB permeability assays, the mean value of the permeability coefficients (Pe) of BBB permeable compounds (propranolol, pyrilamine, memantine, and diphenhydramine) was 960 × 10-6 cm/s, which was clearly distinguishable from that of BBB nonpermeable compounds (sodium fluorescein and Lucifer yellow, 18 × 10-6 cm/s). Collectively, this study successfully developed the hiBBB model, which exhibits essential BBB functionality. Taking into consideration the high availability of the immortalized cells used in the hiBBB model, our results are expected to become an initial step toward the establishment of a useful human BBB model to investigate drug penetration into the human brain.

Inhibition of the Inflammatory Pathway Enhances Both the in Vitro and in Vivo Transfection Activity of Exogenous in Vitro-Transcribed mRNAs Delivered by Lipid Nanoparticles.

While the use of in vitro-transcribed mRNA (IVT-mRNA) in therapeutics is a rapidly expanding area, the transfection of the exogenous IVT-mRNA is accompanied by a risk of immune activation. This immunological defense mechanism suppresses cellular translation process and can reduce transfection efficiency to a considerable extent. In the present study, we investigated the in vitro effects of Integrated Stress Response Inhibitor (ISRIB), and dexamethasone, a steroidal anti-inflammatory drug, on the transfection activity of a lipid nanoparticle (LNP) that was composed of ionizable lipids and IVT-mRNA. In the case of transfection to mouse embryonic fibroblast (MEF) cells, ISRIB mainly enhanced the transfection activity at an early stage of transfection (0-6 h). In contrast, dexamethasone caused an increase in transfection activity at intermediate-late stages of transfection (4-48 h). We also investigated the in vivo effects of dexamethasone using an LNP on that the IVT-mRNA and lipid-conjugated dexamethasone (Dex-Pal) were co-loaded. The intravenous administration of the LNP successfully enhanced the protein expression in a mouse liver by up to 6.6-fold. Collectively, the co-delivery of an anti-inflammatory drug is a promising approach for enhancing transfection efficiency of IVT-mRNA.

Comparison of the hepatic metabolism of triazolam in wild-type andCyp3a-knockout mice for understanding CYP3A-mediated metabolism inCYP3A-humanised mice in vivo.

1. To investigate cytochrome P450 3A (CYP3A)-mediated metabolism in vivo, plasma concentrations of triazolam (TRZ) are often monitored as a CYP3A marker in CYP3A-humanised mice. However, it has not been determined whether plasma concentrations of TRZ after intravenous administration can reflect hepatic CYP3A activity in CYP3A-humanised mice. 2. Firstly, we investigated the pharmacokinetics of TRZ in wild-type and Cyp3a-knockout (Cyp3a-KO) mice. Plasma concentration profiles of TRZ and α-hydroxy (OH) TRZ were very similar in wild-type and Cyp3a-KO mice. On the other hand, AUC of 4-OH TRZ in Cyp3a-KO mice was significantly lower than that in wild-type mice. Pregnenolone 16α-carbonitrile (PCN) decreased the areas under the plasma concentration-time curves (AUCs) of TRZ and α-OH TRZ in both groups. There was no significant effect of PCN on AUC of 4-OH TRZ in Cyp3a-KO mice. 3. Next, we verified that AUC of 4-OH TRZ in CYP3A-humanised mice was higher than that in Cyp3a-KO mice, although the difference was not significant. 4. In conclusion, plasma concentrations of 4-OH TRZ, but not those of TRZ and α-OH TRZ, might reflect hepatic CYP3A activity in mice in vivo. These results provide important insights for in vivo studies using a CYP3A-humanised model.