Transcutaneous Cancer Vaccine Using a Reverse Micellar Antigen Carrier.

Skin dendritic cells (DCs) such as Langerhans cells and dermal dendritic cells have a pivotal role in inducing antigen-specific immunity; therefore, transcutaneous cancer vaccines are a promising strategy to prophylactically prevent the onset of a variety of diseases, including cancers. The largest obstacle to delivering antigen to these skin DC subsets is the barrier function of the stratum corneum. Although reverse micellar carriers are commonly used to enhance skin permeability to hydrophilic drugs, the transcutaneous delivery of antigen, proteins, or peptides has not been achieved to date because of the large molecular weight of drugs. To achieve effective antigen delivery to skin DCs, we developed a novel strategy using a surfactant as a skin permeation enhancer in a reverse micellar carrier. In this study, glyceryl monooleate (MO) was chosen as a skin permeation enhancer, and the MO-based reverse micellar carrier enabled the successful delivery of antigen to Langerhans cells and dermal dendritic cells. Moreover, transcutaneous vaccination with the MO-based reverse micellar carrier significantly inhibited tumor growth, indicating that it is a promising vaccine platform against tumors.

BK channel dysfunction disrupts attention-controlled behaviors and altered perseverative responses in murine instrumental learning.

This study examined the effect of knockout of KCNMA1 gene, coding for the BK channel, on cognitive and attentional functions in mice, with an aim to better understand its implications for human neurodevelopmental disorders. The study used the 3-choice serial reaction time task (3-CSRTT) to assess the learning performance, attentional abilities, and repetitive behaviors in mice lacking the KCNMA1 gene (KCNMA1-/-) compared to wild-type (WT) controls. Results showed no significant differences in learning accuracy between the two groups. However, KCNMA1-/- mice were more prone to omitting responses to stimuli. In addition, when the timing of cue presentation was randomized, the KCNMA1-/- showed premature responses. Notably, these mice also demonstrated a marked reduction in perseverative responses, which include repeated nose-poke behaviors following decisions. These findings highlight the involvement of the KCNMA1 gene in managing attention, impulsivity, and potentially moderating repetitive actions.

Filamin C plays an essential role in the maintenance of the structural integrity of cardiac and skeletal muscles, revealed by the medaka mutant zacro.

Filamin C is an actin-crosslinking protein that is specifically expressed in cardiac and skeletal muscles. Although mutations in the filamin C gene cause human myopathy with cardiac involvement, the function of filamin C in vivo is not yet fully understood. Here we report a medaka mutant, zacro (zac), that displayed an enlarged heart, caused by rupture of the myocardiac wall, and progressive skeletal muscle degeneration in late embryonic stages. We identified zac to be a homozygous nonsense mutation in the filamin C (flnc) gene. The medaka filamin C protein was found to be localized at myotendinous junctions, sarcolemma, and Z-disks in skeletal muscle, and at intercalated disks in the heart. zac embryos showed prominent myofibrillar degeneration at myotendinous junctions, detachment of myofibrils from sarcolemma and intercalated disks, and focal Z-disk destruction. Importantly, the expression of γ-actin, which we observed to have a strong subcellular localization at myotendinous junctions, was specifically reduced in zac mutant myotomes. Inhibition of muscle contraction by anesthesia alleviated muscle degeneration in the zac mutant. These results suggest that filamin C plays an indispensable role in the maintenance of the structural integrity of cardiac and skeletal muscles for support against mechanical stress.

ERK2 contributes to the control of social behaviors in mice.

Signaling through extracellular signal-regulated kinase (ERK) is important in multiple signal transduction networks in the CNS. However, the specific role of ERK2 in in vivo brain functions is not fully understood. Here we show that ERK2 play a critical role in regulating social behaviors as well as cognitive and emotional behaviors in mice. To study the brain function of ERK2, we used a conditional, region-specific, genetic approach to target Erk2 using the Cre/loxP strategy with a nestin promoter-driven cre transgenic mouse line to induce recombination in the CNS. The resulting Erk2 conditional knock-out (CKO) mice, in which Erk2 was abrogated specifically in the CNS, were viable and fertile with a normal appearance. These mice, however, exhibited marked anomalies in multiple aspects of social behaviors related to facets of autism-spectrum disorders: elevated aggressive behaviors, deficits in maternal nurturing, poor nest-building, and lower levels of social familiarity and social interaction. Erk2 CKO mice also exhibited decreased anxiety-related behaviors and impaired long-term memory. Pharmacological inhibition of ERK1 phosphorylation in Erk2 CKO mice did not affect the impairments in social behaviors and learning disabilities, indicating that ERK2, but not ERK1 plays a critical role in these behaviors. Our findings suggest that ERK2 has complex and multiple roles in the CNS, with important implications for human psychiatric disorders characterized by deficits in social behaviors.

Imaging characteristics of hypertrophic pachymeningitis due to ANCA-associated vasculitis.

OBJECTIVE: Hypertrophic pachymeningitis (HP) is a rare disorder that causes localized or diffuse inflammatory fibrosis and thickening of the dura mater. Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV)-related HP is the most frequent form of HP. Otitis media with AAV (OMAAV) patients with HP are associated with higher rates of both ANCA-negative phenotypes and disease-related mortality. However, few studies have reported the imaging characteristics of HP due to AAV/OMAAV. Therefore, we investigated this issue in the present study. METHODS: This retrospective study included patients diagnosed with HP between 2011 and 2020 at our hospital. Age, sex, causative disease, serum C-reactive protein (CRP) level, and MRI data were collected from medical records. We compared the locations of MRI enhancement depending on the causative diseases. RESULTS: Of the 18 included patients with HP (mean age, 64.1 ± 2.6 years; range, 33-77 years), 10 (55.6%) were female, 12 (66.7%) were diagnosed with AAV/OMAAV, four (22.2%) were diagnosed as idiopathic, two (11.1%) were diagnosed with invasive Aspergillus mastoiditis. Eleven (61.1%) had cranial neuropathies. Facial nerve paralysis was common in AAV/OMAAV, while abducent nerve paralysis was common in idiopathic HP. Cranial fossa enhancement was most common presentation in patients with HP, whereas inner acoustic canal (IAC) enhancement was seen only in patients with AAV/OMAAV, while HP involving the cavernous sinus was seen only in patients with idiopathic and mastoiditis. CONCLUSION: HP involving the IAC may be a key factor in diagnosing AAV/OMAAV.

Morphological characterization of cholinergic partition cells: A transmitter-specific tracing study by Cre/lox-dependent viral gene expression.

BACKGROUND: Partition cells are cholinergic interneurons located in lamina VII of the spinal cord. Some partition cells are the source of the cholinergic boutons, known as C-terminals or C-boutons, that modulate the activity of spinal motor neurons. Therefore, partition cells might play an important role in motor control. Previous studies categorized partition cells into three groups (medial, intermediate, and lateral partition cells) according to their distance from the central canal. However, the morphological characteristics of the three groups remain obscure. METHODS: To analyze the morphology of partition cells, we developed an efficient technique for visualization of specific neurons at single-cell level in particular positions using adenovirus vectors and Cre/lox mediated recombination. Cre/lox conditional vectors were injected into the spinal cord of choline acetyltransferase-Cre transgenic mice, and partition cells labeled by green fluorescent protein were reconstructed from histological serial sections at the single-cell level. RESULTS: This technique allowed for the visualization of partition cells at high resolution and revealed that partition cells had various patterns of dendrite orientations and fields. Most of the visualized partition cells had more than 60% of their dendrites located in lamina VII of the spinal cord. Partition cells had dendrites extending into various Rexed's laminae (V, VI, VII, VIII, IX, and X), but none of the cells had dendrites extending dorsal to lamina IV. The dendrites of partition cells terminated both ipsilaterally and bilaterally. We also found that C-terminals on motor neurons may be derived from the middle/outer group of partition cells. CONCLUSIONS: Our results indicated that partition cells have various morphological features of the dendritic pattern and may receive differential inputs. Our results suggested that C-terminals originate not only from medial but also from intermediate/lateral cholinergic partition cells. The present study suggests that intermediate/lateral partition cells modulate activities of motor neurons through C-terminal synapses.

Effect of hormonal therapy on the otoconial changes caused by estrogen deficiency.

Benign paroxysmal positional vertigo (BPPV) is associated with menopause and/or osteopenia. Morphological changes in the otoconial layer have been reported after ovariectomy (OVX). Moreover, hormone replacement therapy decreases BPPV risk. However, knowledge concerning the effect of hormonal therapy on the otoconial changes caused by estrogen deficiency is limited. We aimed to examine the effect of hormonal therapy on otoconial changes caused by estrogen deficiency. We hypothesized that hormonal therapy could reduce otoconial changes caused by OVX. Eight-week-old C57BL/6 mice were divided into four groups: sham operation with implantation of vehicle (sham + v), OVX with implantation of vehicle (OVX + v), OVX with implantation of estradiol (E2) (OVX + E2), and OVX with implantation of raloxifene (RAL) (OVX + RAL) groups. Otoconial layer volume was measured by micro-CT at 4 weeks after OVX or the sham operation. The otic bullae were removed; immunohistochemistry was performed for estrogen receptor alpha and 4-hydroxynonenal. Otoconial layer volume was significantly higher in the OVX + v than in the sham + v group. E2 and RAL significantly reduced these changes in the endometrial layer. The staining of estrogen receptor alpha and 4-hydroxynonenal were stronger in the OVX + v than in the sham + v group but equal in the sham + v, OVX + E2, and OVX + RAL groups. These results indicate that E2 and RAL are effective against morphological changes of the otoconial layer caused by estrogen deficiency via oxidative stress reduction.

Simultaneous detection of T lymphocyte-related antigens (CD4/CD8, CD57, TCRß) with nuclei by fluorescence-based immunohistochemistry in paraffin-embedded human lymph node, liver cancer and stomach cancer.

OBJECTIVE: To develop a method for immunohistochemical staining of three different T lymphocyte antigens (CD4 or CD8, CD57 and TCRß) on the same tissue section and to determine whether tissues have been infiltrated with T lymphocytes expressing these markers. STUDY DESIGN: Commercially available antibodies were tested for immunohistochemical usefulness in a dye-based conventional single-immunostaining method after antigen retrieval on paraffin-embedded human lymph nodes. We searched for the combination of antibodies that could detect T lymphocyte antigens on the same section without any cross-reactivity and that have fluorescent signals robust enough to overcome paraffin autofluorescence. RESULTS: Application of the antigen retrieval technique and the Sudan black B quenching technique enabled staining of paraffin-embedded tissue sections with fluorescent-labeled secondary antibodies. The combination of primary and secondary antibodies that could simultaneously detect the T lymphocyte antigens CD4 or CD8, CD57 and TCRß in histochemical analysis of a paraffin-embedded human lymph node section was established, and was successfully applied to a human tissue section infiltrated with T lymphocytes that express these markers. CONCLUSION: The antibodies listed here would be helpful for histopathologists who wish to investigate T lymphocytes in the paraffin-embedded sections that have accumulated in pathology labs throughout the world.

Association of the prognosis and severity of idiopathic sudden sensorineural hearing loss with cervical ultrasonographic findings.

OBJECTIVE: The aim of this study was to investigate the association of the prognosis and severity of idiopathic sudden sensorineural hearing loss (ISSNHL) with cervical ultrasonographic findings suggestive of cardiovascular risk. METHODS: Seventy-four inpatients with ISSNHL were included in our study. Cervical ultrasonography was performed to evaluate the common carotid artery (CCA), internal carotid artery (ICA), and vertebral artery (VA). The peak systolic velocity, end diastolic velocity, intima-media thickness, pulsatility index (PI), and resistance index (RI) were evaluated. We investigated the relationship of these variables with the severity and prognosis of ISSNHL. RESULTS: ICA-PI, ICA-RI, and CCA-RI were significantly higher in patients with poor hearing prognosis than in those with good prognosis. The variables of VA were not related to the prognosis of ISSNHL. There were no statistically significant differences between ISSNHL severity and cervical ultrasonographic findings. CONCLUSIONS: We found that PI and RI might be prognostic factors for ISSNHL.

Characterization of a Plasmodium falciparum PHISTc protein, PF3D7_0801000, in blood- stage malaria parasites.

During intraerythrocytic development Plasmodium falciparum deploys numerous proteins to support erythrocyte invasion, intracellular growth and development, as well as host immune evasion. Since these proteins are key for parasite intraerythrocytic survival and propagation, they represent attractive targets for antimalarial vaccines. In this study we sought to characterize a member of the PHISTc family of proteins, PF3D7_0801000, as a potential vaccine target. Using the wheat germ cell-free system we expressed the N-terminal region of PF3D7_0801000 (G93-L494, PF3D7_0801000N) and generated specific immune sera. We observed that PF3D7_0801000 localizes in merozoites, and antibodies against PF3D7_0801000N modestly inhibit P. falciparum parasite growth in in vitro culture. Sliding window analysis of the coding sequence revealed that pf3d7_0801000n is relatively conserved among African parasite isolates. Antibody profiles in a malaria-exposed Ugandan population revealed that PF3D7_0801000N is strongly immunoreactive with antibody acquisition increasing with age. Taken together, these findings suggest the need for further evaluation of PF3D7_0801000 for its role in merozoite invasion and utility as an asexual blood-stage vaccine candidate antigen.

Overexpression of the dominant-negative form of myostatin results in doubling of muscle-fiber number in transgenic medaka (Oryzias latipes).

In addition to altering the phenotypes of gene-modified animals, transgenesis also has the potential to facilitate access to the various mechanisms underlying the development and functioning of specific phenotypes and genes, respectively. Myostatin (MSTN) is implicated in double-muscling when mutated in mammals, indicating that MSTN is a negative regulator of skeletal muscle formation. In order to elucidate the role of an MSTN equivalent in fish muscle formation, we created a transgenic medaka strain that expresses dominant-negative MSTN exclusively in skeletal muscle, d-rR-Tg(OlMA1-C315Y-MSTN-hrGFPII-FLAG). The transgenic fish exhibited increased production of skeletal muscle fibers at the adult stage (hyperplasia), although gross muscle mass was not altered. During embryogenesis, ectopic accumulation and misalignment of muscle fibers, possibly due to muscle-fiber hypertrophy, were observed in the transgenic medaka. Our findings suggest that MSTN function is required for regulating the appropriate growth of skeletal muscle in medaka. Unlike in mammals, MSTN loss-of-function failed to induce double-muscling in medaka, despite the highly conserved nature of MSTN function among taxa.

Neurochemical characterization of mouse dorsal root ganglion neurons expressing organic cation transporter 2.

Organic cation transporters (OCTs) are poly-specific carriers for endogenous and exogenous cationic compounds. These are widely distributed in the nervous system and mediate neuronal activities. As antineoplastic cationic drugs accumulate in the dorsal root ganglion (DRG), OCT function has been studied mainly in cultured DRG neurons. However, the histological distribution of OCTs in the DRG is unclear. This study investigated the localization of OCT2 (a member of OCTs) in mouse DRG neurons and determined their histochemical properties. OCT2 expression was found in about 20% of DRG neurons, which were small to medium size. OCT2-expressing neurons were labeled with markers for peptidergic nociceptive (substance P or calcitonin gene-related peptide) and tactile/proprioceptive (neurofilament 200 or tropomyosin receptor kinase B or C) neurons. OCT2 was also expressed in cholinergic DRG neurons identified by choline acetyltransferase promoter-derived Cre expression. In the spinal dorsal horn, OCT2 was distributed in superficial to deep laminae. OCT2 immunoreactivity was punctate in appearance and localized in the nerve terminals of sensory afferents with labeling of neurochemical markers. Our findings suggest that OCT2 as a low-affinity, high-capacity carrier may take up substrates including cationic neurotransmitters and drugs from the extracellular space around cell bodies in DRG neurons.

Formulation Development and Characterization of Nanoemulsion-Based Formulation for Topical Delivery of Heparinoid.

Heparinoid is commonly used for the treatment of superficial thrombophlebitis, a condition wherein inflammation and clotting occurs in the veins below the skin surface. However, stratum corneum is a major barrier that limits the delivery of hydrophilic heparinoid, in and across the skin. The aim of the present study was to develop a nonirritant topical formulation for heparinoid incorporating chemical penetration enhancers and investigate the delivery of heparinoid across the human epidermis using in vitro vertical Franz diffusion cells. The developed oil-in-water nanoemulsions (NEs; NE-1 and NE-2) delivered higher amount of heparinoid (91.58 ± 25.75 µg/sq.cm and 62.67 ± 5.66 µg/sq.cm, respectively) after 72 h compared with the other developed formulations, which in turn also delivered significantly higher amount compared with commercial formulations: cream (1.78 ± 0.07 µg/sq.cm), ointment (9.95 ± 4.41 µg/sq.cm), and gel (0 µg/sq.cm) (p <0.05). Transmission electron microscopy, polarizing light microscopy, and dynamic light scattering studies were performed to characterize the microstructure of these NEs with chemical enhancers. NE-1 was tested to be nonirritant with cell viability greater than 50% and a minimal release of IL-1α by using the "in vitro Epiderm tissue" model. Our results demonstrate that NE formulations represent a potential strategy for providing a localized therapy for the treatment of superficial thrombophlebitis.

PV1, a novel Plasmodium falciparum merozoite dense granule protein, interacts with exported protein in infected erythrocytes.

Upon invasion, Plasmodium falciparum exports hundreds of proteins across its surrounding parasitophorous vacuole membrane (PVM) to remodel the infected erythrocyte. Although this phenomenon is crucial for the parasite growth and virulence, elucidation of precise steps in the export pathway is still required. A translocon protein complex, PTEX, is the only known pathway that mediates passage of exported proteins across the PVM. P. falciparum Parasitophorous Vacuolar protein 1 (PfPV1), a previously reported parasitophorous vacuole (PV) protein, is considered essential for parasite growth. In this study, we characterized PfPV1 as a novel merozoite dense granule protein. Structured illumination microscopy (SIM) analyses demonstrated that PfPV1 partially co-localized with EXP2, suggesting the protein could be a PTEX accessory molecule. Furthermore, PfPV1 and exported protein PTP5 co-immunoprecipitated with anti-PfPV1 antibody. Surface plasmon resonance (SPR) confirmed the proteins' direct interaction. Additionally, we identified a PfPV1 High-affinity Region (PHR) at the C-terminal side of PTP5 where PfPV1 dominantly bound. SIM analysis demonstrated an export arrest of PTP5ΔPHR, a PTP5 mutant lacking PHR, suggesting PHR is essential for PTP5 export to the infected erythrocyte cytosol. The overall results suggest that PfPV1, a novel dense granule protein, plays an important role in protein export at PV.

Differential trafficking of carboxyl isoforms of Ca2+-gated (Slo1) potassium channels.

The pore-forming subunit of the large-conductance Ca(2+)-dependent K(+) (Slo1) channel is encoded by one gene. However, the functional properties of Slo1 channels are diverse in part because of their numerous regulatory mechanisms including posttranslational modification and alternative splicing. In particular, multiple splice variants of the pore-forming subunit have been reported but their significance is only beginning to be elucidated. Here we examined the cell biological properties of the three common C-terminal isoforms that differ in the last 8 (Slo1_ERL and Slo1_VYR) or 61 residues (Slo1_DEC). We found that Slo1_DEC, the longest isoform, shows dramatically reduced surface expression compared to that of Slo1_ERL or Slo1_VYR. Immunocytochemistry revealed that a large fraction of Slo1_DEC remains localized in endoplasmic reticulum (ER). Using a GST fusion protein containing the Slo1_DEC-specific sequence, affinity purification was carried out to isolate interacting proteins. The identified proteins include protein phosphatase 2A (PP2A-A), actin, and tubulin. The PP2A-A interaction is specific to Slo1_DEC and causes a significant reduction of phosphorylation in Slo1_DEC but not Slo1_ERL or Slo1_VYR. The results together support the notion that Slo1_DEC nucleates isoform-specific protein complexes and possesses a cis element(s) for regulating trafficking of the Slo1 channels.

Tumor growth limited to subcutaneous site vs tumor growth in pulmonary site exhibit differential effects on systemic immunities.

To evaluate systemic immunity associated with tumor growth limited to a subcutaneous site versus growth proceeding at multiple tumor sites, we established syngeneic mouse subcutaneous and pulmonary tumor models by local subcutaneous and intravenous injection of colon carcinoma CT26 cells. We found that splenic myeloid-derived suppressor cell (MDSC) levels were significantly increased in the subcutaneous tumor model but not in the pulmonary tumor model. Furthermore, both CD4+ and CD8+ T cells as well as CD4+ Foxp3+ T cells were significantly decreased in the subcutaneous tumor model and were largely unchanged in the pulmonary tumor model. In addition, the subcutaneous model, but not the pulmonary model, displayed a Th1 polarization bias. This bias was characterized by decreased IL-4, IL-9, and IL-10 production, whereas the pulmonary model displayed increased production of IL-10. These results suggest that the mode of tumor development has differential effects on systemic immunity that may, in turn, influence approaches to treatment of cancer patients.

Localization of organic cation transporter 2 (OCT2) in monoaminergic and cholinergic axon terminals of the mouse brain.

Organic cation transporters (OCTs) are low-affinity, high-capacity carriers that mediate sodium-independent transport for biogenic cations, including catecholamine, serotonin, histamine, and choline/acetylcholine. Among them, OCT2 is expressed in neurons of the central nervous system. Although previous studies show OCT2 expression in several populations of cholinergic and monoaminergic neurons, the regional distribution of OCT2 in the brain remains largely unknown. Here we performed immunohistochemical analyses to reveal the distribution of OCT2 throughout the mouse forebrain. OCT2 immunoreactivity was widely distributed, with substantial regional specificity in cortical and subcortical structures including the hippocampus, striatum, and some subdivisions of the amygdala and extended amygdala. Interestingly, OCT2 appeared as punctate, bouton-like labeling in cholinergic, dopaminergic, and serotonergic axon terminals that were co-labeled with presynaptic neurochemical markers. We also co-labeled OCT2 and an anterograde tract-tracer injected into the locus coeruleus, demonstrating that OCT2 was localized to presumptive noradrenergic terminals in the forebrain. Together, our results demonstrated that the polyspecific cation transporter OCT2 is distributed in cholinergic and monoaminergic terminals in various forebrain regions, suggesting that OCT2 could play a role in regulating presynaptic reuptake and recycling of choline and monoamines.

BK channels in microglia are required for morphine-induced hyperalgesia.

Although morphine is a gold standard medication, long-term opioid use is associated with serious side effects, such as morphine-induced hyperalgesia (MIH) and anti-nociceptive tolerance. Microglia-to-neuron signalling is critically involved in pain hypersensitivity. However, molecules that control microglial cellular state under chronic morphine treatment remain unknown. Here we show that the microglia-specific subtype of Ca(2+)-activated K(+) (BK) channel is responsible for generation of MIH and anti-nociceptive tolerance. We find that, after chronic morphine administration, an increase in arachidonic acid levels through the µ-opioid receptors leads to the sole activation of microglial BK channels in the spinal cord. Silencing BK channel auxiliary ß3 subunit significantly attenuates the generation of MIH and anti-nociceptive tolerance, and increases neurotransmission after chronic morphine administration. Therefore, microglia-specific BK channels contribute to the generation of MIH and anti-nociceptive tolerance.

High affinity D- and L-serine transporter Asc-1: cloning and dendritic localization in the rat cerebral and cerebellar cortices.

System asc transporter Asc-1, expressed in the brain, transports D- and L-serine with high affinity. To determine the localization of Asc-1 in the rat brain, we isolated a cDNA for the rat orthologue of Asc-1. The encoded protein designated as rAsc-1 (rat Asc-1) exhibited 98% sequence identity to mouse Asc-1 (mAsc-1). Based on amino acid sequences of rAsc-1 and mAsc-1, two polyclonal antibodies against Asc-1 were generated and used for the immunohistochemical analysis on the cerebral and cerebellar cortices of rats and mice. Asc-1 immunoreactivity was detected in neurons, including cerebellar Purkinje neurons and pyramidal neurons in the neocortex and hippocampus. It was clearly localized in dendrites as well as somata. The localization of Asc-1 in brain suggests the significant contribution of Asc-1 to amino acid mobilization in brains including the synaptic clearance of D-serine and the neuronal uptake of L-serine that is essential for survival and dendrite growth of Purkinje neurons in particular.