A-910823, a squalene-based emulsion adjuvant, induces T follicular helper cells and humoral immune responses via α-tocopherol component.

Background: Adjuvants are chemical or biological materials that enhance the efficacy of vaccines. A-910823 is a squalene-based emulsion adjuvant used for S-268019-b, a novel vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is currently in clinical development. Published evidence has demonstrated that A-910823 can enhance the induction of neutralizing antibodies against SARS-CoV-2 in humans and animal models. However, the characteristics and mechanisms of the immune responses induced by A-910823 are not yet known. Methods and Results: To characterize A-910823, we compared the adaptive immune response profile enhanced by A-910823 with that of other adjuvants (AddaVax, QS21, aluminum salt-based adjuvants, and empty lipid nanoparticle [eLNP]) in a murine model. Compared with other adjuvants, A-910823 enhanced humoral immune responses to an equal or greater extent following potent T follicular helper (Tfh) and germinal center B (GCB) cell induction, without inducing a strong systemic inflammatory cytokine response. Furthermore, S-268019-b containing A-910823 adjuvant produced similar results even when given as a booster dose following primary administration of a lipid nanoparticle-encapsulated messenger RNA (mRNA-LNP) vaccine. Preparation of modified A-910823 adjuvants to identify which components of A-910823 play a role in driving the adjuvant effect and detailed evaluation of the immunological characteristics induced by each adjuvant showed that the induction of humoral immunity and Tfh and GCB cell induction in A-910823 were dependent on α-tocopherol. Finally, we revealed that the recruitment of inflammatory cells to the draining lymph nodes and induction of serum cytokines and chemokines by A-910823 were also dependent on the α-tocopherol component. Conclusions: This study demonstrates that the novel adjuvant A-910823 is capable of robust Tfh cell induction and humoral immune responses, even when given as a booster dose. The findings also emphasize that α-tocopherol drives the potent Tfh-inducing adjuvant function of A-910823. Overall, our data provide key information that may inform the future production of improved adjuvants.

Preparation and pharmaceutical properties of Hangeshashinto oral ointment and its safety and efficacy in Syrian hamsters with 5-fluorouracil-induced oral mucositis.

Chemotherapy-induced oral mucositis (COM) is a common adverse effect of cancer chemotherapy. Several clinical studies reported that repetitive use of mouthwashes containing 2.5-6.25% Hangeshashinto (HST), a Kampo formula, relieves COM, but the effect is insufficient. To solve this problem, we produced an oral ointment of 12% HST extract (considered quantitatively equivalent to 20% commercially available HST), which will increase the local concentrations of its active ingredients and prolong the contact time with COM. In this study, we evaluated the pharmaceutical properties (spreadability and stability) of HST oral ointment. In addition, its safety (oral mucosal irritation) and therapeutic effects on 5-fluorouracil-induced oral mucositis were evaluated in male Syrian hamsters. The HST ointment showed good spreadability and stability for more than 8 weeks at 4 °C. In the oral mucosal irritation test, topical application of HST ointment (0.2 g) three times per day for 14 days had no adverse effect on the oral mucosa of hamsters. In hamsters treated with 5-fluorouracil (60 mg/kg) twice, COM was induced by a submucosal injection of 5% acetic acid into the cheek pouch. When HST ointment (50 µg) was topically applied to the mucositis area once per day for 12 days, the area and macroscopic score of mucositis were significantly decreased, and the depth of the wound tended to be reduced compared with the lactose ointment-treated control animals. These findings suggest that HST oral ointment shows good properties in spreadability, stability, and safety, and elicits a therapeutic effect in an animal model of COM.

Effect of riboflavin deficiency on development of the cerebral cortex in Slc52a3 knockout mice.

Riboflavin transporter 3 (RFVT3), encoded by the SLC52A3 gene, is important for riboflavin homeostasis in the small intestine, kidney, and placenta. Our previous study demonstrated that Slc52a3 knockout (Slc52a3-/-) mice exhibited neonatal lethality and metabolic disorder due to riboflavin deficiency. Here, we investigated the influence of Slc52a3 gene disruption on brain development using Slc52a3-/- embryos. Slc52a3-/- mice at postnatal day 0 showed hypoplasia of the brain and reduced thickness of cortical layers. At embryonic day 13.5, the formation of Tuj1+ neurons and Tbr2+ intermediate neural progenitors was significantly decreased; no significant difference was observed in the total number and proliferative rate of Pax6+ radial glia. Importantly, the hypoplastic phenotype was rescued upon riboflavin supplementation. Thus, it can be concluded that RFVT3 contributes to riboflavin homeostasis in embryos and that riboflavin itself is required during embryonic development of the cerebral cortex in mice.

Disruption of Slc52a3 gene causes neonatal lethality with riboflavin deficiency in mice.

Homeostasis of riboflavin should be maintained by transporters. Previous in vitro studies have elucidated basic information about riboflavin transporter RFVT3 encoded by SLC52A3 gene. However, the contribution of RFVT3 to the maintenance of riboflavin homeostasis and the significance in vivo remain unclear. Here, we investigated the physiological role of RFVT3 using Slc52a3 knockout (Slc52a3-/-) mice. Most Slc52a3-/- mice died with hyperlipidemia and hypoglycemia within 48 hr after birth. The plasma and tissue riboflavin concentrations in Slc52a3-/- mice at postnatal day 0 were dramatically lower than those in wild-type (WT) littermates. Slc52a3-/- fetuses showed a lower capacity of placental riboflavin transport compared with WT fetuses. Riboflavin supplement during pregnancy and after birth reduced neonatal death and metabolic disorders. To our knowledge, this is the first report to indicate that Rfvt3 contributes to placental riboflavin transport, and that disruption of Slc52a3 gene caused neonatal mortality with hyperlipidemia and hypoglycemia owing to riboflavin deficiency.

Prevention of antipsychotic-induced hyperglycaemia by vitamin D: a data mining prediction followed by experimental exploration of the molecular mechanism.

Atypical antipsychotics are associated with an increased risk of hyperglycaemia, thus limiting their clinical use. This study focused on finding the molecular mechanism underlying antipsychotic-induced hyperglycaemia. First, we searched for drug combinations in the FDA Adverse Event Reporting System (FAERS) database wherein a coexisting drug reduced the hyperglycaemia risk of atypical antipsychotics, and found that a combination with vitamin D analogues significantly decreased the occurrence of quetiapine-induced adverse events relating diabetes mellitus in FAERS. Experimental validation using mice revealed that quetiapine acutely caused insulin resistance, which was mitigated by dietary supplementation with cholecalciferol. Further database analysis of the relevant signalling pathway and gene expression predicted quetiapine-induced downregulation of Pik3r1, a critical gene acting downstream of insulin receptor. Focusing on the phosphatidylinositol 3-kinase (PI3K) signalling pathway, we found that the reduced expression of Pik3r1 mRNA was reversed by cholecalciferol supplementation in skeletal muscle, and that insulin-stimulated glucose uptake into C2C12 myotube was inhibited in the presence of quetiapine, which was reversed by concomitant calcitriol in a PI3K-dependent manner. Taken together, these results suggest that vitamin D coadministration prevents antipsychotic-induced hyperglycaemia and insulin resistance by upregulation of PI3K function.

In vivo evaluation of neutron capture therapy effectivity using calcium phosphate-based nanoparticles as Gd-DTPA delivery agent.

PURPOSE: A more immediate impact for therapeutic approaches of current clinical research efforts is of major interest, which might be obtained by developing a noninvasive radiation dose-escalation strategy, and neutron capture therapy represents one such novel approach. Furthermore, some recent researches on neutron capture therapy have focused on using gadolinium as an alternative or complementary for currently used boron, taking into account several advantages that gadolinium offers. Therefore, in this study, we carried out feasibility evaluation for both single and multiple injections of gadolinium-based MRI contrast agent incorporated in calcium phosphate nanoparticles as neutron capture therapy agent. METHODS: In vivo evaluation was performed on colon carcinoma Col-26 tumor-bearing mice irradiated at nuclear reactor facility of Kyoto University Research Reactor Institute with average neutron fluence of 1.8 × 10(12) n/cm(2). Antitumor effectivity was evaluated based on tumor growth suppression assessed until 27 days after neutron irradiation, followed by histopathological analysis on tumor slice. RESULTS: The experimental results showed that the tumor growth of irradiated mice injected beforehand with Gd-DTPA-incorporating calcium phosphate-based nanoparticles was suppressed up to four times higher compared to the non-treated group, supported by the results of histopathological analysis. CONCLUSION: The results of antitumor effectivity observed on tumor-bearing mice after neutron irradiation indicated possible effectivity of gadolinium-based neutron capture therapy treatment.

[Utility of organotypic raphe slice cultures to investigate the effects of psychotropic drugs on the function of serotoninergic neurons].

A number of behavioral, neurochemical and electrophysiological studies have emphasized the importance of the serotonergic neurons in the pathophysiology of psychiatric disorders and the therapeutic actions of psychotropics. However, no in vitro serotonergic culture systems have successfully analyzed the long-term effects of psychotropics or the neural interaction between serotonergic and excitatory/inhibitory neurons. Recently, we have established rat organotypic raphe slice cultures, which have functional serotonergic neurons with the ability to release 5-HT in response to stimulation and to reuptake 5-HT through serotonin transporter and retain neural and synaptic functions. Here, we show the following results in the raphe slice cultures: 1) acute and sustained treatments with 3,4-methylenedioxymethamphetamine induce the 5-HT efflux via serotonin transporter and AMPA receptor-mediated exocytotic 5-HT release, respectively; 2) sustained treatment with antidepressants enhances the exocytotic 5-HT release, which is dependent on AMPA receptor stimulation, but not on desensitization of 5-HT(1A/1B) autoreceptors; 3) the augmentation therapy of an atypical antipsychotic, olanzapine, with antidepressants is caused by the decrease in the raphe inhibitory GABAergic tone through 5-HT6 receptor antagonism. Our findings suggest that the raphe slice cultures are suitable for analyzing the neural and molecular mechanisms underlying the efficacy of psychotropics in vitro.

Trauma-specific insults to the cochlear nucleus in the rat.

The effect of acoustic overstimulation on the neuronal number of the cochlear nucleus (CN) was investigated by using unbiased stereological methods in rats. We found that, after 9 weeks of recovery, neurons in the anteroventral cochlear nucleus (AVCN) degenerated, whereas those in the posteroventral and dorsal cochlear nuclei (PVCN and DCN) were preserved. The noise trauma induced near complete loss of the outer hair cells throughout the cochlea, and the inner hair cells were preserved only in the more apical regions. This pattern of selective loss of AVCN neurons in this study was different from trauma induced by auditory deafferentation by mechanical compression of auditory neurons. In contrast to noise trauma, mechanical compression caused loss of neurons in the PVCN and DCN. After 5 weeks of recovery from mechanical compression, there was no loss of inner or outer hair cells. These findings indicate that auditory deprivation, induced by different experimental manipulations, can have strikingly different consequences for the central auditory system. We hypothesized that AVCN neuronal death was induced by excitotoxic mechanisms via AMPA-type glutamate receptors and that excitatory neuronal circuits developed after acoustic overstimulation protected the PVCN and DCN against neuronal death. The results of the present study demonstrate that hearing loss from different etiologies will cause different patterns of neuronal degeneration in the CN. These findings are important for enhancing the performance of cochlear implants and auditory brainstem implants, because diverse types of hearing loss can selectively affect neuronal degeneration of the CN.

Localization of septin proteins in the mouse cochlea.

Septins are a family of GTP binding proteins that are well conserved in eukaryotic species except plants. Septins contribute to the lateral compartmentalization of membranes, cortical rigidity, and the regulation of membrane trafficking by associating with membrane lipids, actin, and microtubules. The organ of Corti in the cochlea has pivotal roles in auditory perception and includes two kinds of highly polarized cells, hair and supporting cells, both of which are rich in actin and microtubules. To identify the roles of septins in the cochlea, we analyzed the localization of three septin proteins, septin 4 (SEPT4), septin 5 (SEPT5), and septin 7 (SEPT7) that are abundantly expressed in brain tissues, and also examined auditory functions of Sept4 and Sept5 null mice. SEPT4, SEPT5, and SEPT7 were expressed in inner and outer pillar cells and Deiters' cells but the distribution patterns of each protein in Deiters' cells were different. SEPT4 and SEPT7 were expressed in the phalangeal process where SEPT5 was not detected. In addition to these cells SEPT5 and SEPT7 were co-localized with presynaptic vesicles of efferent nerve terminals. Only SEPT7 was expressed in the cochlea at embryonic stages. Although expression patterns of septin proteins suggested their important roles in the function of the cochlea, both Sept4 and Sept5 null mice had similar auditory functions to their wild type littermates. Immunohistochemical analysis of Sept4 null mice showed that compensatory expression of SEPT5 in the phalangeal process of Deiters' cells may have caused functional compensation of hearing ability in Sept4 null mice.

Involvement of NOX1/NADPH oxidase in morphine-induced analgesia and tolerance.

The involvement of reactive oxygen species (ROS) in morphine-induced analgesia and tolerance has been suggested, yet how and where ROS take part in these processes remains largely unknown. Here, we report a novel role for the superoxide-generating enzyme NOX1/NADPH oxidase in the regulation of analgesia and acute analgesic tolerance. In mice lacking Nox1 (Nox1(-/Y)), the magnitude of the analgesia induced by morphine was significantly augmented. More importantly, analgesic tolerance induced by repeated administration of morphine was significantly suppressed compared with that in the littermates, wild-type Nox1(+/Y). In a membrane fraction obtained from the dorsal spinal cord, no difference was observed in morphine-induced [(35)S]GTPγS-binding between the genotypes, whereas morphine-stimulated GTPase activity was significantly attenuated in Nox1(-/Y). At 2 h after morphine administration, a significant decline in [(35)S]GTPγS-binding was observed in Nox1(+/Y) but not in Nox1(-/Y). No difference in the maximal binding and affinity of [(3)H]DAMGO was observed between the genotypes, but the translocation of protein kinase C isoforms to the membrane fraction following morphine administration was almost completely abolished in Nox1(-/Y). Finally, the phosphorylation of RGS9-2 and formation of a complex by Gαi2/RGS9-2 with 14-3-3 found in morphine-treated Nox1(+/Y) were significantly suppressed in Nox1(-/Y). Together, these results suggest that NOX1/NADPH oxidase attenuates the pharmacological effects of opioids by regulating GTPase activity and the phosphorylation of RGS9-2 by protein kinase C. NOX1/NADPH oxidase may thus be a novel target for the development of adjuvant therapy to retain the beneficial effects of morphine.

A novel technique for inducing local inner ear damage.

With significant development of mouse genomics and the availability of transgenic and knockout mice, the mouse will be the preferred animal model for inner ear research. However, few studies have used mice as experimental animals for examination of hair cell degeneration, because of their relative resistance to ototoxic agents and difficulties in surgical treatment. This study presents a model for induction of apoptotic cell death in sensory epithelia of the mouse inner ear using injection of neomycin into the posterior semicircular canal. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay revealed that local application of neomycin produced sufficient induction of apoptotic cell death in both auditory and vestibular epithelia over a definite time course. Supplementation of the general caspase-inhibitor significantly reduced induction of TUNEL-positive cells, indicating caspase-dependency of apoptotic cell death observed in the present model. In addition, the approach to the posterior semicircular canal was an easy technique, and sham-operation induced no significant induction of TUNEL-positive cells. This model, hence, enables the use of various genetic tools in studies for mechanisms of hair cell apoptosis.