Annual two-dose tetanus toxoid vaccination induces protective humoral immunity to all age groups of rhesus macaques.

A tetanus outbreak occurred during 2014-2015 in the rhesus macaques reared in an open enclosure in our facility. As the soil of the facility was suspected to be contaminated with Clostridium tetani spores, there was a risk of further tetanus occurring among the macaques. To protect them from tetanus, a tetanus toxoid vaccination was recommended; however, the vaccinated elderly animals might not be effectively protected due to insufficient humoral immune responses. Hence, we evaluated the dynamics of antibody responses among rhesus macaques of all age groups vaccinated with two-dose tetanus toxoid at a 1-year interval during a 3-year follow-up study. The vaccination developed anti-tetanus toxin-specific antibodies in animals of all age groups, the antibody levels peaked 1 year after the second vaccination, and the peak levels decreased with age. However, the levels among elderly individuals (aged ≥13 years) were still higher than the threshold level, which was supposed to protect them from tetanus development. Although the rhesus macaques in our facility had a risk of occasional exposure to the spores due to the outbreak, no incidence of tetanus has ever occurred to date. These results indicate that the vaccination protocol is effective in protecting not only younger but also older animals from tetanus.

Vaccination with short-term-cultured autologous PBMCs efficiently activated STLV-1-specific CTLs in naturally STLV-1-infected Japanese monkeys with impaired CTL responses.

A small proportion of human T-cell leukemia virus type-1 (HTLV-1)-infected individuals develop adult T-cell leukemia/lymphoma, a chemotherapy-resistant lymphoproliferative disease with a poor prognosis. HTLV-1-specific cytotoxic T lymphocytes (CTLs), potential anti-tumor/virus effectors, are impaired in adult T-cell leukemia/lymphoma patients. Here, using Japanese monkeys naturally infected with simian T-cell leukemia/T-lymphotropic virus type-1 (STLV-1) as a model, we demonstrate that short-term-cultured autologous peripheral blood mononuclear cells (PBMCs) can serve as a therapeutic vaccine to activate such CTLs. In a screening test, STLV-1-specific CTL activity was detectable in 8/10 naturally STLV-1-infected monkeys. We conducted a vaccine study in the remaining two monkeys with impaired CTL responses. The short-term-cultured PBMCs of these monkeys spontaneously expressed viral antigens, in a similar way to PBMCs from human HTLV-1 carriers. The first monkey was subcutaneously inoculated with three-day-cultured and mitomycin C (MMC)-treated autologous PBMCs, and then boosted with MMC-treated autologous STLV-1-infected cell line cells. The second monkey was inoculated with autologous PBMC-vaccine alone twice. In addition, a third monkey that originally showed a weak STLV-1-specific CTL response was inoculated with similar autologous PBMC-vaccines. In all three vaccinated monkeys, marked activation of STLV-1-specific CTLs and a mild reduction in the STLV-1 proviral load were observed. Follow-up analyses on the two monkeys vaccinated with PBMCs alone indicated that STLV-1-specific CTL responses peaked at 3-4 months after vaccination, and then diminished but remained detectable for more than one year. The significant reduction in the proviral load and the control of viral expression were associated with CTL activation but also diminished 6 and 12 months after vaccination, respectively, suggesting the requirement for a booster. The vaccine-induced CTLs in these monkeys recognized epitopes in the STLV-1 Tax and/or Envelope proteins, and efficiently killed autologous STLV-1-infected cells in vitro. These findings indicated that the autologous PBMC-based vaccine could induce functional STLV-1-specific CTLs in vivo.

Neutralization of hepatitis B virus with vaccine-escape mutations by hepatitis B vaccine with large-HBs antigen.

Although the current hepatitis B (HB) vaccine comprising small-HBs antigen (Ag) is potent and safe, attenuated prophylaxis against hepatitis B virus (HBV) with vaccine-escape mutations (VEMs) has been reported. We investigate an HB vaccine consisting of large-HBsAg that overcomes the shortcomings of the current HB vaccine. Yeast-derived large-HBsAg is immunized into rhesus macaques, and the neutralizing activities of the induced antibodies are compared with those of the current HB vaccine. Although the antibodies induced by the current HB vaccine cannot prevent HBV infection with VEMs, the large-HBsAg vaccine-induced antibodies neutralize those infections. The HBV genotypes that exhibited attenuated neutralization via these vaccines are different. Here, we show that the HB vaccine consisting of large-HBsAg is useful to compensate for the shortcomings of the current HB vaccine. The combined use of these HB vaccines may induce antibodies that can neutralize HBV strains with VEMs or multiple HBV genotypes.

Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro.

Human T-cell leukemia virus type 1 (HTLV-1) causes serious and intractable diseases in some carriers after infection. The elimination of infected cells is considered important to prevent this onset, but there are currently no means by which to accomplish this. We previously developed "virotherapy", a therapeutic method that targets and kills HTLV-1-infected cells using a cytolytic recombinant vesicular stomatitis virus (rVSV). Infection with rVSV expressing an HTLV-1 primary receptor elicits therapeutic effects on HTLV-1-infected envelope protein (Env)-expressing cells in vitro and in vivo. Simian T-cell leukemia virus type 1 (STLV-1) is closely related genetically to HTLV-1, and STLV-1-infected Japanese macaques (JMs) are considered a useful HTLV-1 surrogate, non-human primate model in vivo. Here, we performed an in vitro drug evaluation of rVSVs against STLV-1 as a preclinical study. We generated novel rVSVs encoding the STLV-1 primary receptor, simian glucose transporter 1 (JM GLUT1), with or without an AcGFP reporter gene. Our data demonstrate that these rVSVs specifically and efficiently infected/eliminated the STLV-1 Env-expressing cells in vitro. These results indicate that rVSVs carrying the STLV-1 receptor could be an excellent candidate for unique anti-STLV-1 virotherapy; therefore, such antivirals can now be applied to STLV-1-infected JMs to determine their therapeutic usefulness in vivo.

The Novel PKC Activator 10-Methyl-Aplog-1 Combined with JQ1 Induced Strong and Synergistic HIV Reactivation with Tolerable Global T Cell Activation.

The presence of latent human immunodeficiency virus (HIV) reservoirs is a major obstacle to a cure. The "shock and kill" therapy is based on the concept that latent reservoirs in HIV carriers with antiretroviral therapy are reactivated by latency-reversing agents (LRAs), followed by elimination due to HIV-associated cell death or killing by virus-specific cytotoxic T lymphocytes. Protein kinase C (PKC) activators are considered robust LRAs as they efficiently reactivate latently infected HIV. However, various adverse events hamper the intervention trial of PKC activators as LRAs. We found in this study that a novel PKC activator, 10-Methyl-aplog-1 (10MA-1), combined with an inhibitor of bromodomain and extra-terminal domain motifs, JQ1, strongly and synergistically reactivated latently infected HIV. Notably, higher concentrations of 10MA-1 alone induced the predominant side effect, i.e., global T cell activation as defined by CD25 expression and pro-inflammatory cytokine production in primary CD4+ T lymphocytes; however, JQ1 efficiently suppressed the 10MA-1-induced side effect in a dose-dependent manner. Considering the reasonable accessibility and availability of 10MA-1 since the chemical synthesis of 10MA-1 requires fewer processes than that of bryostatin 1 or prostratin, our results suggest that the combination of 10MA-1 with JQ1 may be a promising pair of LRAs for the clinical application of the "shock and kill" therapy.

Frequent horizontal and mother-to-child transmission may contribute to high prevalence of STLV-1 infection in Japanese macaques.

BACKGROUND: Simian T-cell leukemia virus type 1 (STLV-1) is disseminated among various non-human primate species and is closely related to human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Notably, the prevalence of STLV-1 infection in Japanese macaques (JMs) is estimated to be > 60%, much greater than that in other non-human primates; however, the mechanism and mode of STLV-1 transmission remain unknown. The aim of this study is to examine the epidemiological background by which STLV-1 infection is highly prevalent in JMs. RESULTS: The prevalence of STLV-1 in the JMs rearing in our free-range facility reached up to 64% (180/280 JMs) with variation from 55 to 77% among five independent troops. Anti-STLV-1 antibody titers (ABTs) and STLV-1 proviral loads (PVLs) were normally distributed with mean values of 4076 and 0.62%, respectively, which were mostly comparable to those of HTLV-1-infected humans. Our initial hypothesis that some of the macaques might contribute to frequent horizontal STLV-1 transmission as viral super-spreaders was unlikely because of the absence of the macaques exhibiting abnormally high PVLs but poor ABTs. Rather, ABTs and PVLs were statistically correlated (p < 0.0001), indicating that the increasing PVLs led to the greater humoral immune response. Further analyses demonstrated that the STLV-1 prevalence as determined by detection of the proviral DNA was dramatically increased with age; 11%, 31%, and 58% at 0, 1, and 2 years of age, respectively, which was generally consistent with the result of seroprevalence and suggested the frequent incidence of mother-to-child transmission. Moreover, our longitudinal follow-up study indicated that 24 of 28 seronegative JMs during the periods from 2011 to 2012 converted to seropositive (86%) 4 years later; among them, the seroconversion rates of sexually matured (4 years of age and older) macaques and immature macaques (3 years of age and younger) at the beginning of study were comparably high (80% and 89%, respectively), suggesting the frequent incidence of horizontal transmission. CONCLUSIONS: Together with the fact that almost all of the full-adult JMs older than 9 years old were infected with STLV-1, our results of this study demonstrated for the first time that frequent horizontal and mother-to-child transmission may contribute to high prevalence of STLV-1 infection in JMs.

IgA regulates the composition and metabolic function of gut microbiota by promoting symbiosis between bacteria.

Immunoglobulin A (IgA) promotes health by regulating the composition and function of gut microbiota, but the molecular requirements for such homeostatic IgA function remain unknown. We found that a heavily glycosylated monoclonal IgA recognizing ovalbumin coats Bacteroides thetaiotaomicron (B. theta), a prominent gut symbiont of the phylum Bacteroidetes. In vivo, IgA alters the expression of polysaccharide utilization loci (PUL), including a functionally uncharacterized molecular family provisionally named Mucus-Associated Functional Factor (MAFF). In both mice and humans, MAFF is detected predominantly in mucus-resident bacteria, and its expression requires the presence of complex microbiota. Expression of the MAFF system facilitates symbiosis with other members of the phylum Firmicutes and promotes protection from a chemically induced model of colitis. Our data reveal a novel mechanism by which IgA promotes symbiosis and colonic homeostasis.

Effect of circulatory system response to motor control in one-sided contractions.

PURPOSE: The purpose of this study was to clarify the effect one-sided skeletal muscle contraction has on the circulatory system, spinal α-motoneuron excitability, and somatosensory-system-evoked potential. METHOD: Nine healthy males maintained tension at 10, 20, and 30% of maximal voluntary contraction in static gripping in right hand. Heart rate, ln high frequency (HF), blood pressure (BP), F-wave, and somatosensory-evoked potential (SEP) were recorded during gripping task. BP, F-wave and SEP were recorded from left hand (contralateral side from contracting side). RESULT AND CONCLUSION: There were significant main effects of contractions strength on heart rate (0%: 68.2 ± 6.8 bpm, 10%: 67.6 ± 7.4 bpm, 20%: 69.7 ± 8.5 bpm, 30%: 73.7 ± 9.3 bpm, F3.24=9.18, P < 0.01), systolic BP (0%: 127.7 ± 15 mmHg, 10%: 136.2 ± 13.5 mmHg, 20%: 136.2 ± 13.5 mmHg, 30%: 140.0 ± 17.1 mmHg, F3.24=23.93, P < 0.01), diastolic BP (0%: 69.3 ± 8.5 mmHg, 10%: 76.9 ± 11.1 mmHg, 20%: 79.9 ± 12.5 mmHg, 30%: 86.2 ± 14 mmHg, F3.24=17.09, P < 0.01), and F-wave appearance rate (0%: 29.7 ± 15.6%, 10%: 39.3 ± 20.5%, 20%: 47.5 ± 22.9%, 30%: 55.2 ± 21.8%, F3.24=14.04, P < 0.01). For the ln HF (0%: 5.9 ± 0.6, 10%: 6.3 ± 0.9, 20%: 6.3 ± 1.3, 30%: 6.0 ± 1.0, F3.24=2.43, P = 0.08), F-wave latency (0%: 29.6 ± 1.7 ms, 10%: 26.9 ± 2.1 ms, 20%: 26.5 ± 3.6 ms, 30%: 26.9 ± 2.3 ms, F3.24=0.11, P = 0.96), F-wave amplitude (0%: 2.0 ± 0.9%, 10%: 2.2 ± 0.9%, 20%: 2.3 ± 0.7%, 30%: 2.8 ± 1.1%, F3.24=2.80, P = 0.06), and N20 amplitude (0%: 3.9 ± 1.7 µV, 10%: 3.7 ± 1.7 µV, 20%: 3.9 ± 1.7 µV, 30%: 3.9 ± 1.8 µV, F3.24=0.61, P = 0.62), between the conditions. We conclude that regulation of the circulatory system and motor system has a limited effect on sensory input.

Clinical Diagnosis of Mendelian Disorders Using a Comprehensive Gene-Targeted Panel Test for Next-Generation Sequencing.

BACKGROUND: Genetic diagnoses provide beneficial information to patients and families. However, traditional genetic diagnoses are often difficult even for experienced clinicians and require recognition of characteristic patterns of signs or symptoms to guide targeted genetic testing for the confirmation of diagnoses. Next-generation sequencing (NGS) is a powerful genetic diagnostic tool. However, whole-genome and whole-exome sequencing (WES) are expensive, and the interpretation of results is difficult. Hence, target gene capture sequencing of gene panels has recently been applied to genetic diagnoses. Herein, we demonstrate that targeted sequencing approaches using gene panel testing are highly efficient for the diagnosis of Mendelian disorders. METHODS: NGS using TruSight one gene panel was performed in 17 families and 20 patients, and we developed a bioinformatic pipeline at our institution for detecting mutations. RESULTS: We detected causative mutations in 6 of 17 (35%) families. In particular, 11 (65%) families had syndromic diagnosis and 6 (35%) had no syndromic diagnosis before NGS testing. The number of positive diagnoses was 5 of 11 (45%) in the syndromic group and were 1 of 6 (17%) among patients of the no syndromic diagnosis group. CONCLUSION: Diagnostic yields in the present study were higher than in previous reports of genetic and chromosomal tests and WES. The present comprehensive gene-targeted panel test is a powerful diagnostic tool for Mendelian disorders.

Adipogenic effects of piperlonguminine in 3T3-L1 cells and plasma concentrations of several amide constituents from Piper chaba extracts after treatment of mice.

In our previous study, piperlonguminine from the fruit of Piper chaba was reported to promote adipogenesis in 3T3-L1 cells like the peroxisome proliferator-activated receptor-γ (PPARγ) agonist, troglitazone. In the present study, the mode of action of piperlonguminine in cells was examined. Piperlonguminine increased mRNA levels of adiponectin, glucose transporter 4, and fatty acid-binding protein (aP2). It also increased mRNA levels of PPARγ2 but, unlike troglitazone, piperlonguminine did not activate PPARγ directly in a nuclear receptor cofactor assay. Analyses of plasma from mice treated with piperlonguminine, piperine, and retrofractamide A, and an extract of the fruit, showed that concentrations of piperlonguminine were higher than those of piperine and retrofractamide A, and that the "area-under-the-curve" of piperine increased following in vivo administration of the extract.

Genetic similarity of circulating and small intestinal virus at the end stage of acute pathogenic simian-human immunodeficiency virus infection.

To understand the pathogenicity of acquired immune deficiency syndrome (AIDS), it is important to clarify where, when and how the virus replicates in the body of infected individuals. To identify the major virus replication site at the end stage of SHIV infection, we investigated the systemic tissues of SHIV-infected monkeys that developed AIDS-like disease. We quantified proviral DNA, and compared the mutation patterns of the viruses in various systemic tissues and in peripheral blood through phylogenetic analysis of the full genome sequence. We found that the amounts of proviral DNA detected in internal tissues were higher than those in peripheral blood mononuclear cells. In the sequence and phylogenetic tree analyses, the mutation patterns of the viruses in each tissue were generally different. However, the mutation pattern of the viruses in the jejunum and mesenteric lymph node were most similar to that of plasma viral RNA among the tissues examined in all three monkeys. In two of the three monkeys, which were euthanized earlier, viruses in the jejunum and mesenteric lymph node occupied the root position of the phylogenetic tree. Furthermore, in these tissues, more than 50% of SHIV-expressing cells were identified as macrophages based on co-expression of CD68. These results suggest that macrophages of the small intestine and/or mesenteric lymph node are the major virus production site at the end stage of SHIV infection of macaques.

Medicinal flowers. XXXIII. Anti-hyperlipidemic and anti-hyperglycemic effects of chakasaponins I-III and structure of chakasaponin IV from flower buds of Chinese tea plant (Camellia sinensis).

Effects of principal saponins, chakasaponins I-III, from the flower buds of Camellia sinensis cultivated in Fujian province, China on plasma triglyceride (TG) and glucose levels in olive oil or sucrose-loaded mice were examined. Chakasaponins I-III at 50 and 100 mg/kg significantly inhibited increases in plasma TG and glucose levels. Furthermore, they prevented gastric emptying, suggesting that the former inhibitory effect is partly dependent on the inhibition of gastric emptying. In addition, the chemical structure of a new acylated oleanane-type triterpene oligoglycoside, chakasaponin IV, was elucidated on the basis of chemical and physicochemical evidence.

Successful treatment for sympathetic storms in a patient with neuro-Behçet's disease.

Sympathetic storms (SyS) are characterized by hyperactivity of autonomic functions, resulting in episodes of hyperthermia, hypertension, tachycardia, and hyperhidrosis. We show here a patient with neuro-Behçet's disease (NBD) complicated by SyS. Although SyS is well known to occur with brain tumors, trauma, and hydrocephalus, this is the first report to show that SyS is a manifestation of central nervous system involvement in a patient with NBD. High concentrations of norepinephrine (NE) and IL-8 in cerebrospinal fluid reflected the activity of SyS. The patient's symptoms showed almost complete improvement after treatment with corticosteroids and intravenous cyclophosphamide. Also, the concentrations of NE and IL-8 were decreased to normal levels. An awareness of the potential for SyS and adequate immunosuppressant therapy are of importance when dealing with patients with NBD.

Expression of respiratory chain enzyme mRNA and the morphological properties of mitochondria in the masseter muscles of klotho mutant mice.

The activity of respiratory chain enzymes in a rat's masseter muscle changes as the animal ages; however, there is little information about the RNA transcript levels of mitochondrial enzymes in klotho mutant mice as they age. We measured the activities of NADH-ferricyanide oxidoreductase and NADH-O2 oxidoreductase, and the RNA transcript levels of NADH dehydrogenase, the mitochondrial isoform of ND1, the nuclear isoforms of the 51 kDa and 75 kDa subunits of Complex I, the nuclear isoform of cytochrome c, and the mitochondrial isoform of beta subunits of ATPase (Complex V). In addition, we measured the RNA transcript levels of catalase (CAT) and superoxide dismutase (SOD), which are associated with antioxidant proteins. Moreover, we measured ATP concentrations using a luciferin-luciferase assay, and we determined the amount of cytochrome c associated with mitochondria in both klotho mutant mice and wild-type mice. However, the mRNA levels of cytochrome c and Complex V components, the mRNA levels of CAT, SOD, and apoptosis-inducing factor (Aifm), and the protein level of cytochrome c remained constant as klotho mutant mice aged from 5 weeks to 7 weeks. In wild-type mice, these components (except for those of Complex I) increased over time. NADH-ferricyanide oxidoreductase and NADH-O2 oxidoreductase activities decreased in klotho mutant mice as they aged from 5 weeks to 7 weeks. A few large mitochondria were scattered between myofibrils, and 7-week-old klotho mutant mice displayed an increased number of irregular mitochondria with fewer cristae. Our results indicate that the klotho protein plays a role in the diminished functional adaptability of enzymes in the masseter muscle of klotho mutant mice throughout the aging process.

Distribution of slow muscle fiber of muscle spindle in postnatal rat masseter muscle.

We investigated the properties of the muscle spindle in the masseter muscle at an immunohistochemical level in rats fed for 6 weeks. Slow myosin heavy chain (MyHC) isoforms were measured and intrafusal fibers in the muscle spindle were studied to determine the relationship between the superficial and deep regions of rat masseter muscle after alternated feeding pattern. However, muscle spindles were found in both regions, mainly in the deep region of the posterior superficial region of masseter muscle. The total number of the slow fiber in the intrafusal fiber and number of muscle spindle in the deep region were high from 5 to 8 weeks old in spite of various dimensions of data such as diameter and the compositions of the intrafusal fiber. The relationship of the protein expression of slow MyHC in the two regions at 5 weeks old reversed five weeks later (10 weeks old). This period is an important stage because the mastication system in masseter muscle with muscle spindle may be changed during the alternated feeding pattern of suckling to mastication. The changes may be a marker of the feeding system and of the control by the tension receptor of muscle spindle in this stage of masseter muscle after postnatal development.