Long-term protective immunity induced by an adjuvant-containing live-attenuated AIDS virus.

The use of an adjuvant in vaccination is thought to be effective for enhancing immune responses to various pathogens. We genetically constructed a live attenuated simian human immunodeficiency virus (SHIV) to express the adjuvant molecule Ag85B (SHIV-Ag85B). SHIV-Ag85B could not be detected 4 weeks after injection in cynomolgus macaques, and strong SHIV-specific T cell responses were induced in these macaques. When the macaques in which SHIV-Ag85B had become undetectable were challenged with pathogenic SHIV89.6P at 37 weeks after SHIV-Ag85B had become undetectable, SHIV89.6P was not detected after the challenge. Eradication of SHIV89.6P was confirmed by adoptive transfer experiments and CD8-depletion studies. The SHIV-Ag85B-inoculated macaques showed enhancement of Gag-specific monofunctional and polyfunctional CD8+ T cells in the acute phase of the pathogenic SHIV challenge. The results suggest that SHIV-Ag85B elicited strong sterile immune responses against pathogenic SHIV and that it may lead to the development of a vaccine for AIDS virus infection.

A Case of Topical Ofloxacin-Induced Otomycosis and Literature Review.

The prevalence of fungal otitis externa, or otomycosis, has been increasing in recent decades. Fungi may act as primary pathogens in this condition, or they may occur as secondary infections after prolonged ototopical treatment with antibiotics, which alters the flora of the external auditory canal (EAC) and enables overgrowth of its fungal inhabitants. We report here a case of otomycosis by Candida parapsilosis, Malassezia obtusa, and Malassezia furfur as a secondary infection following prolonged otic ofloxacin treatment. To the best of our knowledge, although isolation of C. parapsilosis and M. furfur from the EAC is not uncommon, the recovery of M. obtusa has not yet been reported. We also conducted a literature review of the searchable data on PubMed concerning the isolation of Malassezia species from the human EAC.

A murine model of eosinophilic chronic rhinosinusitis using the topical application of a vitamin D3 analog.

BACKGROUND: Eosinophilic chronic rhinosinusitis (ECRS) is a chronic inflammatory disease, characterized by eosinophilic infiltration, T-helper type 2 (Th2-type) response, and olfactory dysfunction. A master regulator of Th2-type inflammation, thymic stromal lymphopoietin (TSLP), is important for basophil activation. TSLP-elicited basophils are a key factor in the pathogenesis of ECRS. METHODS: In order to elucidate the mechanisms of ECRS in humans, we aimed to establish a murine model of ECRS based on TSLP production in response to the topical application of MC903 (a vitamin D3 analog) and the subsequent TSLP-induced basophil activation. Histological analyses were performed to assess immune cell infiltration into the nasal mucosa and to explore the impact of eosinophilic inflammation on the olfactory epithelium. The status of Th2-type inflammation was evaluated by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). RESULTS: Eosinophils, basophils, and M2 macrophages increased significantly in the nasal mucosa of the mice treated with MC903 and ovalbumin (OVA), compared to those treated with OVA alone or the controls. Quantitative real-time PCR and ELISA revealed elevated expression of interleukin (IL)-4, IL-5, IL-13, TSLP, the chemokine CCL11, and CCL24 in the nasal mucosa of the ECRS mice. In parallel, thinned olfactory epithelium and decreased mature olfactory sensory neurons were observed in the ECRS mice. CONCLUSIONS: Our model of ECRS displayed Th2-type inflammation in the sinonasal region, including both eosinophil infiltration and basophil infiltration. Additionally, olfactory epithelium turned out to be affected by eosinophilic inflammation. These features are consistent with the characteristics of the human ECRS.

Metabolism of Odorant Molecules in Human Nasal/Oral Cavity Affects the Odorant Perception.

In this study, we examined the mode of metabolism of food odorant molecules in the human nasal/oral cavity in vitro and in vivo. We selected 4 odorants, 2-furfurylthiol (2-FT), hexanal, benzyl acetate, and methyl raspberry ketone, which are potentially important for designing food flavors. In vitro metabolic assays of odorants with saliva/nasal mucus analyzed by gas chromatography mass spectrometry revealed that human saliva and nasal mucus exhibit the following 3 enzymatic activities: (i) methylation of 2-FT into furfuryl methylsulfide (FMS); (ii) reduction of hexanal into hexanol; and (iii) hydrolysis of benzyl acetate into benzyl alcohol. However, (iv) demethylation of methyl raspberry ketone was not observed. Real-time in vivo analysis using proton transfer reaction-mass spectrometry demonstrated that the application of 2-FT and hexanal through 3 different pathways via the nostril or through the mouth generated the metabolites FMS and hexanol within a few seconds. The concentration of FMS and hexanol in the exhaled air was above the perception threshold. A cross-adaptation study based on the activation pattern of human odorant receptors suggested that this metabolism affects odor perception. These results suggest that some odorants in food are metabolized in the human nasal mucus/saliva, and the resulting metabolites are perceived as part of the odor quality of the substrates. Our results help improve the understanding of the mechanism of food odor perception and may enable improved design and development of foods in relation to odor.

Low CT Attenuation Values of Sinonasal Benign Tumours Relative to the Brainstem Identify Schwannomas.

OBJECTIVES: On computed tomography (CT), sinonasal schwannoma displays as a soft-tissue mass without any distinctive features. Our aim was to define the radiological criteria for distinguishing schwannoma from other sinonasal benign tumours. METHODS: We retrospectively identified consecutive patients who were pathologically diagnosed with benign sinonasal tumours between 2007 and 2016. CT attenuation values were compared between benign tumours and the brainstem. The utilities of demographic factors, clinical factors, and CT parameters for predicting the CT attenuation values of the brainstem were analysed by univariate and multivariate regression. RESULTS: Of the 111 identified cases of benign tumours, the CT attenuation values of tumours and the brainstem were analysed in 36 cases (schwannoma, 4 cases; inverted papilloma, 26; juvenile nasopharyngeal angiofibroma, 3; cavernous haemangioma, 3). The CT attenuation values of the schwannomas were significantly lower than in the brainstem, while those of the other tumours were significantly higher than in the brainstem. No factors affected the CT attenuation values of the brainstem. CONCLUSION: Low CT attenuation values of sinonasal benign tumours relative to the brainstem could distinguish schwannomas from other benign tumours.

Influence of the location of nasal polyps on olfactory airflow and olfaction.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) often results in decreased olfaction. In this study, we examined the relationship between nasal polyp location and olfactory airflow and odorant transport changes using virtual nasal polyp models at different locations and computational fluid dynamics (CFD) analysis. We also compared olfactory airflow and olfaction between patients with nasal polyps at different locations using CFD analysis and an olfactory test. METHODS: Nasal computed tomography images were used to generate a normal model and 4 virtual nasal polyp models based on polyp locations, including the olfactory region (all-olfactory model), the region anterior to the olfactory region (preolfactory model), the middle meatus (middle-meatus model), and the superior meatus (superior-meatus model). Various airflow parameters were compared between these models and a normal model without polyps. We then performed a similar comparison between the 3-dimensional (3D) reconstruction models of patients with nasal polyps, and retrospectively investigated the correlation between olfaction and nasal polyp location in those patients. RESULTS: Virtual nasal polyp analysis revealed dispersion of olfactory airflow in the all-olfactory model. Olfactory airflow and odorant transport showed maximum decrease in the preolfactory model and a slight decrease in the superior-meatus model. Olfactory airflow by polyps was further decreased by blockade of the olfactory airflow inlet than of the outlet. The findings obtained by patients corresponded well to those of the virtual polyp analysis. CONCLUSION: Olfactory airflow and olfaction are differentially affected by nasal polyp location. This finding is important for planning polyp-removal surgeries from the perspective of improving patient olfaction.

Pulsatile tinnitus and carotid artery dissection.

Carotid artery dissection is an uncommon entity associated with head and neck pain, partial Horner's syndrome, amaurosis fugax, and brain ischemia, which may all occur in isolation or in combination. Herein, we report a rare case of cervical artery dissection in which pulsatile tinnitus was the only reported symptom. A 38-years-old man attended our hospital with a 4-days history of left side pulsatile tinnitus which began after stumbling. He had no other symptom. MRA showed luminal stenosis with pseudo lumen of the internal carotid artery. The patient was diagnosed with left internal carotid artery dissection and treated with antihypertensive therapy accordingly. After 2 months, the stenosis and tinnitus spontaneously resolved.

Intravenous olfactory test latency correlates with improvement in post-infectious olfactory dysfunction.

CONCLUSION: This cohort study showed that onset latency in the intravenous olfactory test (IVO) may help predict when olfaction in patients with post-infectious olfactory dysfunction (PIOD) improves. OBJECTIVES: To identify factors that predict the olfactory improvement period in patients with PIOD. METHODOLOGY/PRINCIPAL: All consecutive patients presenting with PIOD in 1994-2014 who were followed up for 2 years were identified retrospectively. The ability of demographic/clinical factors (age, sex, body mass index, presence/absence of allergic rhinitis, treatment/non-treatment with herbal medicines, patient dependence on herbal medicine treatment, presence/absence of diabetes mellitus, and smoking status) and olfactory test factors (response/no response and onset latency and duration in the IVO test, and detection and recognition scores on the T&T olfactory test) to predict the olfactory improvement period (defined respectively as the time from PIOD onset or olfactory testing to the first self-report of olfaction improvement) was analyzed by univariate and multivariate regression. RESULTS: Of the 187 PIOD patients, the prognostic ability of demographic/clinical factors was analyzed in 65. None predicted the olfactory improvement period. Of the 65 patients, 20 did not respond in the IVO test. In the remaining 45 patients, onset latency (but not the other olfactory test factors) was a significant prognosticator of olfactory improvement period (R2=0.24, p = 0.003).

Poly-2-methoxyethylacrylate-coated cardiopulmonary bypass circuit can reduce transfusion of platelet products compared to heparin-coated circuit during aortic arch surgery.

Several coating techniques for extracorporeal circulation have been developed to reduce the systemic inflammatory response during cardiopulmonary bypass (CPB). We compared the clinical effectiveness and biocompatibility of poly-2-methoxyethylacrylate (PMEA)- and heparin-coated CPB circuits in total aortic arch replacement (TAR) with the prolonged use of the bypass technique. Twenty patients who underwent elective TAR were divided randomly into two equal groups: group P (n = 10) to use PMEA-coated circuits and group H (n = 10) to use heparin-coated circuits. Clinical outcomes, hematological variables, and acute phase inflammatory response were analyzed perioperatively. Demographic, CPB, and clinical outcome data were similar for both groups. Hemoglobin and platelet count showed similar time-course curves. However, the amount of platelet products transfused intraoperatively was significantly larger in group H (group P 26.0 ± 7.0 units; group H 33.0 ± 6.7 units, p = 0.04). Total protein, and albumin levels were significantly higher in group P during and after the operation (total protein, p = 0.04; albumin, p = 0.02). The use of PMEA-coated circuit is associated with retainment of perioperative plasma proteins levels and may help to reduce transfusion of platelet products in TAR in comparison with the heparin-coated circuit.

Analysis of antibody response by temperature-sensitive measles vaccine strain in the cotton rat model.

Measles virus (MeV) vaccine strain, AIK-C, is temperature sensitive (ts), which is thought to be associated with attenuation of virus pathogenicity. In this study, replication and antibody response were examined in cotton rats using viruses carrying different forms of the P gene, which is responsible for the ts phenotype of strain AIK-C and its parental Edmonston strain. When cotton rats were inoculated intranasally, ts viruses neither replicated in lungs, nor reproducibly generated an antibody response. When inoculated intramusculary (i.m.), however, ts strains raised an antibody titer in all animals. This response was not observed when ultraviolet-inactivated virus was used. ts virus, inoculated i.m., was recovered from cotton rat drainage lymph nodes. These results suggest that ts virus, inoculated i.m., could replicate in the cotton rat, presumably at the superficial lymph node, and induce an antibody response. Therefore, cotton rats can serve as a small-animal model for investigating immune responses to safer ts vaccine, as well as recombinant vaccine using AIK-C as a vector for protection against other infectious agents.

Characterization of lectin isolated from Momordica charantia seed as a B cell activator.

Lectin isolated from the seeds of Momordica charantia (MCL) is a galactose-specific glycoprotein. To investigate the effects of MCL on cell activation, we analyzed the responses of BALB/c splenocytes, thymocytes, T cells and B cells on MCL stimulation. Proliferation assays showed that MCL selectively stimulates the B cell subset of splenocytes (p<0.05) in a dose and time dependent manner and that this activation proceeds without the involvement of T cells. Flow cytometric analysis revealed that the fluorescein isothiocyanate (FITC)-labeled MCL binds to B cells, which was inhibited by specific sugars, including galactose. Mouse immunoglobulin (Ig) was able to inhibit MCL-induced proliferation of mouse B cells, suggesting MCL stimulates B cell activation via membrane Ig in the B cell surface. Moreover, after 96-h co-culture, MCL triggered splenocytes to produce a large amount of non-specific IgM in culture supernatants (p<0.01). Additionally, MCL was shown to up-regulate the cell activation marker CD86, in a B cell subpopulation distinct from that affected by LPS. These data suggest that MCL is a T cell-independent B cell activator and a polyclonal Ig inducer, and provide further information on the immunomodulatory effect of MCL.

[Development of oral vaccines].

In the increasing crisis of pandemic of infectious diseases all over the world in recent years, it is the most necessary to develop readily available vaccines even in developing countries. Since many pathogens establish their initial infections through the mucosal surface in our bodies, the induction of mucosal immune responses by vaccines are thought to be important for the prevention of infectious diseases through mucosal site. Oral administration of vaccines has abilities to elicit mucosal immune responses at mucosal tissues with various advantages such as easy skill for administration, less stressful for vaccine recipients and safer than systemic injection. Here, we show our novel strategies for inducing mucosal immune responses by oral vaccine administration.

[Innovation of vaccine adjuvants].

The enhancement of immune responses to vaccine antigen by adjuvants is critical for prevention of infectious disease. Here, we summarized the current status of adjuvant development and adjuvant categories like mineral salts, oil emulsion, and microorganism-derived adjuvants. Our resent study suggested that Ag85B of mycobacteria, which cross-reacts among mycobacteria species, elicits helper T-cell type 1 (Th1) immune responses as a novel adjuvant. These responses were enhanced in mice sensitized by BCG before vaccination. Since most humans have been sensitized by spontaneous infections or by vaccination with mycobacteria, these findings indicate that Ag85B is a promising adjuvant for enhancing Th1 immune responses of vaccine candidates. The study on the mechanisms of adjuvanticity will improve the development of novel vaccine adjuvants for human use.

Immunoadjuvant activity of crude lectin extracted from Momordica charantia seed.

The aim of this study is to investigate the immunoadjuvant activity of the crude Momordica charantia lectin (crMCL) extracted from seed using beta-galactosidase (beta-gal) as the model antigen. BALB/c mice were injected intramuscularly with beta-gal alone or beta-gal + crMCL for up to four immunizations at two-week intervals. After administration of 2 doses, the IgG-specific titer to beta-gal was significantly higher in mice in the beta-gal + crMCL group than in that from the animals from the beta-gal alone group, while it was about the same in both groups after 1 dose. Our data suggest that crMCL may help raise antibodies under the prime and boost administration regimen and could be a potent vaccine adjuvant.

A genetically engineered live-attenuated simian-human immunodeficiency virus that co-expresses the RANTES gene improves the magnitude of cellular immunity in rhesus macaques.

Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper (Th) type-1 responses against HIV-1. To evaluate the adjuvant effects of RANTES against HIV vaccine candidate in SHIV-macaque models, we genetically engineered a live-attenuated SHIV to express the RANTES gene (SHIV-RANTES) and characterized the virus's properties in vivo. After the vaccination, the plasma viral loads were same in the SHIV-RANTES-inoculated monkeys and the parental nef-deleted SHIV (SHIV-NI)-inoculated monkeys. SHIV-RANTES provided some immunity in monkeys by remarkably increasing the antigen-specific CD4+ Th cell-proliferative response and by inducing an antigen-specific IFN-gamma ELISpot response. The magnitude of the immunity in SHIV-RANTES-immunized animals, however, failed to afford greater protection against a heterologous pathogenic SHIV (SHIV-C2/1) challenge compared to control SHIV-NI-immunized animals. SHIV-RANTES immunized monkeys, elicited robust cellular CD4+ Th responses and IFN-gamma ELISpot responses after SHIV-C2/1 challenge. These findings suggest that the chemokine RANTES can augment vaccine-elicited, HIV-specific CD4+ T cell responses.

In vitro and in vivo stability of plasmids in attenuated Salmonella enterica serovar typhimurium used as a carrier of DNA vaccine is associated with its replication origin.

The ability of live attenuated Salmonella enterica serovar typhimurium (S. typhimurium) as a carrier of DNA vaccine was evaluated using model plasmid encoding beta-galactosidase (beta-Gal) and BALB/c mice. We constructed pBRCMVbeta, beta-Gal expression apparatus having a replication origin from low copy pBR322. Comparison of the plasmid stability showed that pBRCMVbeta remained stable in Salmonella even after oral administration, while pUC-based pCMVbeta tended to be lost quickly. However, titers for beta-Gal specific IgG in sera did not significantly increase in mice orally administered S. typhimurium harboring pBRCMVbeta. These data suggest that the stability of plasmid in S. typhimurium is associated with its replication origin. Further studies are required to scientifically establish this methodology.

Construction and in vitro characterization of a chimeric simian and human immunodeficiency virus with the RANTES gene.

Chimeric simian-human immunodeficiency virus (SHIV) containing the env gene of HIV-1 infects macaque monkeys and provides basic information that is useful for the development of HIV-1 vaccines. Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper type-1 responses against HIV-1. With the final goal of testing the adjuvant effects of RANTES in SHIV-macaque models, we constructed a SHIV having the RANTES gene (SHIV-RANTES) and characterized its properties in vitro. SHIV-RANTES replicated both in human and monkey T cell lines. Along with SHIV-RANTES replication, RANTES was detected in the supernatant of human and monkey cell cultures, at maximal levels of 98.5 and 4.1 ng/ml, respectively. A flow cytometric analysis showed that the expressed RANTES down-modulated CC-chemokine receptor 5 (CCR5) on PM1 cells, which was restored by adding anti-RANTES antibody. UV-irradiated culture supernatants from the SHIV-RANTES-infected cells suppressed replication of CCR5-tropic HIV-1 BaL in PM-1 cells. Differentiating real-time RT-PCR showed that pre-infection of SHIV-RANTES in C8166 cells expressing CCR5 suppressed the replication of HIV-1 BaL. Biological activity of the expressed RANTES and the inserted RANTES gene in SHIV-RANTES remained stable after 10 passages. These results suggest that SHIV-RANTES is worth testing in macaque models.

Induction of immune response in macaque monkeys infected with simian-human immunodeficiency virus having the TNF-alpha gene at an early stage of infection.

TNF-alpha has been implicated in the pathogenesis of, and the immune response against, HIV-1 infection. To clarify the roles of TNF-alpha against HIV-1-related virus infection in an SHIV-macaque model, we genetically engineered an SHIV to express the TNF-alpha gene (SHIV-TNF) and characterized the virus's properties in vivo. After the acute viremic stage, the plasma viral loads declined earlier in the SHIV-TNF-inoculated monkeys than in the parental SHIV (SHIV-NI)-inoculated monkeys. SHIV-TNF induced cell death in the lymph nodes without depletion of circulating CD4(+) T cells. SHIV-TNF provided some immunity in monkeys by increasing the production of the chemokine RANTES and by inducing an antigen-specific proliferation of lymphocytes. The monkeys immunized with SHIV-TNF were partly protected against a pathogenic SHIV (SHIV-C2/1) challenge. These findings suggest that TNF-alpha contributes to the induction of an effective immune response against HIV-1 rather than to the progression of disease at the early stage of infection.

Characterization of vpr vector constructed from chimeric simian and human immunodeficiency virus.

Chimeric simian and human immunodeficiency viruses (SHIVs) are useful tool for investigating AIDS pathogenesis and for development of vaccine. We constructed a SHIV-vpr vector (designated as SHIV-3sj) by replacing vpr region with restriction enzyme sites. SHIV-3sj was designed to express inserted gene along with its viral replication. Five cytokine genes were inserted into SHIV-3sj, and ability of viral replication and expression of the inserted genes were examined. The short insert including RANTES and IL-5 resulted in the successful expression from SHIV-3sj, while the construct having longer genes including IL-2, IL-6 and IL-12p35 failed to become replication competent. These results suggest that the length of the insert is an important factor for the replication ability of SHIV-3sj vector.

Trithallium hydrogen bis(sulfate), Tl(3)H(SO(4))(2), in the super-ionic phase by X-ray powder diffraction.

The structure of trithallium hydrogen bis(sulfate), Tl(3)H(SO(4))(2), in the super-ionic phase has been analyzed by Rietveld analysis of the X-ray powder diffraction pattern. Atomic parameters based on the isotypic Rb(3)H(SeO(4))(2) crystal in space group R3m in the super-ionic phase were used as the starting model, because it has been shown from the comparison of thermal and electric properties in Tl(3)H(SO(4))(2) and M(3)H(SO(4))(2) type crystals (M = Rb, Cs or NH(4)) that the room-temperature Tl(3)H(SO(4))(2) phase is isostructural with the high-temperature R3m-symmetry M(3)H(SO(4))(2) crystals. The structure was determined in the trigonal space group R3m and the Rietveld refinement shows that an hydrogen-bond O-H...O separation is slightly shortened compared with O-H...O separations in isotypic M(3)H(SeO(4))(2) crystals. In addition, it was found that the distortion of the SO(4) tetrahedra in Tl(3)H(SO(4))(2) is less than that in isotypic crystals.