1270 nm near-infrared light as a novel vaccine adjuvant acts on mitochondrial photoreception in intradermal vaccines.

With the development of laser technology in the 1960s, a technique was developed to inject intradermal vaccines immediately after irradiating the skin with laser light to elicit an adjuvant effect, referred to as "laser adjuvant." We have been investigating the mechanism of laser adjuvant in influenza mouse models using noninvasive continuous-wave (CW) near-infrared (NIR) light mainly at a wavelength of 1064 nm, and have shown that the production of reactive-oxygen-species (ROS) in the skin and mast cells in the skin tissue plays an important role in the laser adjuvant effect. The new wavelength of 1270 nm NIR light is characterized by its ability to elicit the same vaccine adjuvant effect as other wavelengths at a lower energy, and may be suitable for clinical applications. In this study, we investigated the physiological activity of CW1270 nm NIR light in mast cells, its biological activity on mouse skin, and the durability of the vaccine adjuvant effect in influenza vaccine mouse models. We show that irradiation of mast cells with 1270 nm NIR light produced ROS and ATP, and irradiation of isolated mitochondria also produced ATP. In mouse skin, the relative expression levels of chemokine mRNAs, such as Ccl2 and Ccl20, were increased by irradiation with 1270 and 1064 nm NIR light at minimum safe irradiance. However, the relative expression of Nfkb1 was increased at 1064 nm, but not at 1270 nm. Serum anti-influenza IgG antibody titers increased early after immunization with 1064 nm, whereas with 1270 nm, there was not only an early response of antibody production but also persistence of antibody titers over the medium- to long-term. Thus, to our knowledge, we show for the first time that 1270 nm NIR light induces ROS and ATP production in mitochondria as photoreceptors, initiating a cascade of laser adjuvant effects for intradermal vaccines. Additionally, we demonstrate that there are wavelength-specific variations in the mechanisms and effects of laser adjuvants. In conclusion, CW1270 nm NIR light is expected to be clinically applicable as a novel laser adjuvant that is equivalent or superior to 1064 nm NIR light, because it can be operated at low energy and has a wavelength-specific adjuvant effect with medium- to long-lasting antibody titer.

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.

Chikungunya Fever in Japan Imported from the Caribbean Islands.

A 53-year-old Japanese woman who was working as a volunteer in the Commonwealth of Dominica in the Caribbean islands presented with a high-grade fever and severe incapacitating generalized arthralgia. The Asian genotype of the chikungunya virus was confirmed using reverse transcription-PCR and serology, based on the presence of a specific neutralization titer and immunoglobulin M antibodies. She was diagnosed with post-chikungunya chronic arthritis based on persistence of her polyarthritis for 3 months and the presence of rheumatoid factor, immunoglobulin G-rheumatoid factor, and matrix metalloproteinase-3. Chikungunya virus should be considered as a causative pathogen in travelers returning from Caribbean islands. Clinicians should consider chikungunya fever in the differential diagnosis of patients who complain of chronic arthritis and have a history of travel to an endemic area.

A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification.

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64 °C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 10(3) cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 10(3) cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness.

PURE-LAMP Procedure for the Diagnosis of Extrapulmonary Tuberculosis: A Case Series.

Although the polymerase chain reaction is effective for the diagnosis of extrapulmonary tuberculosis (EPTB), it is typically unavailable in resource-limited situations. In contrast, the loop-mediated isothermal amplification (LAMP) assay is a relatively cost-effective and accessible method. Additionally, when combined with the procedure for ultra-rapid extraction (PURE) kit, which enables simple DNA extraction, LAMP can detect Mycobacterium tuberculosis in sputum within 1.5 hours using a simple procedure. In this study, we investigated the utility of the PURE-LAMP technique to diagnose three cases of EPTB and showed that it may potentially be a valuable tool for the diagnosis of EPTB.

Chronic Chagas disease with advanced cardiac complications in Japan: Case report and literature review.

Due to the unprecedented recent increases in global migration, Chagas disease has become a global health threat and its epidemiology has drastically changed. Here we describe the first case in Japan of benznidazole treatment for chronic Chagas disease characterized by advanced cardiac complications. A 55-year-old Japanese-Brazilian woman who had previously presented with chronic heart failure was diagnosed as having Chagas disease and treated with benznidazole to prevent aggravation of her cardiac complications. However, benznidazole administration was stopped on day 56 due to severe drug-induced peripheral neuritis. Sixteen months later, her serologic test for Trypanosoma cruzi is still positive and she is being followed regularly by cardiology. Despite an estimated prevalence of over 4000 cases in Japan, only a few cases of Chagas disease have been reported. A Medline search revealed only 7 cases identified between 1995 and 2014 in Japan: in 6 cases, complications of chronic Chagas disease were apparent at the time of presentation, and sudden death occurred in 2 of these cases due to cardiac complications. This clinical case and literature review re-emphasize the urgent need to establish a surveillance network and improve the diagnostic methods and treatment framework for Chagas disease in Japan.

Loop-mediated isothermal amplification with the Procedure for Ultra Rapid Extraction kit for the diagnosis of pneumocystis pneumonia.

Loop-mediated isothermal amplification (LAMP) is an innovative molecular technique requiring only a heating device and isothermal conditions to amplify a specific target gene. The results of current microscopic diagnostic tools for pneumocystis pneumonia are not sufficiently consistent for detecting infection with a low-density of Pneumocystis jirovecii. Although polymerase chain reaction (PCR) is highly sensitive, it is not suitable for resource-limited facilities. LAMP is a potential diagnostic replacement for PCR in such settings but a critical disadvantage of DNA extraction was still remained. Therefore, we employed the Procedure for Ultra Rapid Extraction (PURE) kit, which uses a porous material, to isolate the DNA from clinical samples in a simple way in combination with previously reported LAMP procedure for diagnosing PCP. The detection limit of the PURE-LAMP method applied to artificial bronchoalveolar lavage fluid samples was 100 copies/tube, even with the use of massive blood-contaminated solutions. In addition, we concluded the diagnostic procedure within 1 h without the need for additional equipment. PURE-LAMP coupled with suitable primers for specific pathogens has good potential for diagnosing various infectious diseases.

Development of antimicrobial biomaterials produced from chitin-nanofiber sheet/silver nanoparticle composites.

BACKGROUND: Chitin nanofibers sheets (CNFSs) with nanoscale fiber-like surface structures are nontoxic and biodegradable biomaterials with large surface-to-mass ratio. CNFSs are widely applied as biomedical materials such as a functional wound dressing. This study aimed to develop antimicrobial biomaterials made up of CNFS-immobilized silver nanoparticles (CNFS/Ag NPs). MATERIALS AND METHODS: CNFSs were immersed in suspensions of Ag NPs (5.17 ± 1.9 nm in diameter; mean ± SD) for 30 min at room temperature to produce CNFS/Ag NPs. CNFS/Ag NPs were characterized by transmission electron microscopy (TEM) and then tested for antimicrobial activities against Escherichia (E.) coli, Pseudomonas (P.) aeruginosa, and H1N1 influenza A virus, three pathogens that represent the most widespread infectious bacteria and viruses. Ultrathin sectioning of bacterial cells also was carried out to observe the bactericidal mechanism of Ag NPs. RESULTS: The TEM images indicated that the Ag NPs are dispersed and tightly adsorbed onto CNFSs. Although CNFSs alone have only weak antimicrobial activity, CNFS/Ag NPs showed much stronger antimicrobial properties against E. coli, P. aeruginosa, and influenza A virus, with the amount of immobilized Ag NPs onto CNFSs. CONCLUSIONS: Our results suggest that CNFS/Ag NPs interacting with those microbes exhibit stronger antimicrobial activities, and that it is possible to apply CNFS/Ag NPs as anti-virus sheets as well as anti-infectious wound dressings.

The Direct Boil-LAMP method: a simple and rapid diagnostic method for cutaneous leishmaniasis.

Needle biopsy is widely used for the diagnosis of cutaneous leishmaniasis (CL) to obtain specimens for histology and culture. However, the use of such invasive procedures causes discomfort, requires technical expertise, and carries risks of bleeding and iatrogenic infection. Therefore, developing substitutive non-invasive diagnostic tools for CL will help reduce the risk of secondary infection and the exposure of both infected individuals and medical professionals. Here we employed loop-mediated isothermal amplification and boiled swab samples (Direct Boil-LAMP method) from CL model mice to develop a simple and rapid diagnostic method for CL. The detection limit of this procedure was 1.0×10(3)parasites/mL. Accordingly, this substitutive diagnostic method should prove useful for mass screening. In addition, we discuss the potential advantages of using it, particularly in endemic regions where medical resources are limited.

Successful diagnosis of tuberculous lymphadenitis by loop-mediated isothermal amplification of cutaneous samples from an ulcerated surface lesion: a case report.

INTRODUCTION: Tuberculous lymphadenitis is the most frequent form of extrapulmonary tuberculous. Although nucleic acid amplification assays such as polymerase chain reaction have recently become mainstream techniques for diagnosing tuberculous lymphadenitis, they are still not routinely performed in developing countries because of their high costs and complicated procedures. CASE PRESENTATION: We describe a case of tuberculous lymphadenitis in a 79-year-old Japanese man who had been on continuous hemodialysis for end-stage renal disease. We employed loop-mediated isothermal amplification and the procedure for ultrarapid extraction to develop a fast and easy-to-perform procedure for diagnosing tuberculous lymphadenitis. CONCLUSIONS: The commercially available loop-mediated isothermal amplification assay kit and a rapid purification procedure enabled us to identify and amplify a Mycobacterium tuberculosis-specific gene within just 1.5 hours.

Clinical presentation and diagnosis of toxoplasmic encephalitis in Japan.

Distinguishing life-threatening toxoplasmic encephalitis (TE) from brain lymphoma in patients with acquired immunodeficiency syndrome (AIDS) may be difficult. Empiric anti-toxoplasmosis treatment is often initiated because of the reluctance in performing brain biopsies, which may delay the diagnosis and treatment of brain lymphoma in Japan. In this study, we retrospectively examined the clinical characteristics of 13 AIDS patients with TE in Japan, including magnetic resonance imaging and thallium 201 (201TI) single photon emission computed tomography (SPECT) findings, cerebral spinal fluid analysis, serology, and polymerase chain reaction (PCR) results. All patients improved on anti-toxoplasmosis treatment. Of the 11 patients who underwent serological testing, 6 (55%) had a positive serological result. Of the 7 patients who underwent PCR testing, 3 (42.9%) had a positive PCR result. Nine of 11 patients with TE (81.8%) had multiple lesions. Analysis of the sites of TE lesions did not reveal a difference in site predilection between TE and brain lymphoma. Uptake was negative in all 9 patients who underwent 201Tl SPECT. The study findings suggest that toxoplasma serostatus and PCR may be used to discriminate TE from brain lymphoma. No focal accumulation of 201TI is strongly suggestive of TE in patients with AIDS in Japan.

Mother-to-child transmission of congenital Chagas disease, Japan.

We report a patient with congenital Chagas disease in Japan. This report reemphasizes the role of neglected and emerging tropical diseases in the era of globalization. It also indicates the need for increased vigilance for detecting Chagas disease in non-disease-endemic countries.

Preparation of size-controlled silver nanoparticles and chitosan-based composites and their anti-microbial activities.

We previously reported a simple method for the preparation of size-controlled spherical silver nanoparticles (Ag NPs) generated by autoclaving a mixture of silver-containing glass powder and glucose. The particle size is regulated by the glucose concentration, with concentrations of 0.25, 1.0 and 4.0 wt% glucose providing small (3.48 ± 1.83 nm in diameter), medium (6.53 ± 1.78 nm) and large (12.9 ± 2.5 nm) particles, respectively. In this study, Ag NP/chitosan composites were synthesized by mixing each of these three Ag NP suspensions with a 75% deacetylated (DAc) chitosan suspension (pH 5.0) at room temperature. The Ag NPs were homogeneously dispersed and stably embedded in the chitosan matrices. The Ag NP/chitosan composites were obtained as yellow or brown flocs. It was estimated that approximately 60, 120 and 360 µg of the small, medium and large Ag NPs, respectively, were maximally embedded in 1 mg of chitosan. The bactericidal and anti-fungal activities of the Ag NP/chitosan composites increased as the amount of Ag NPs in the chitosan matrix increased. Furthermore, smaller Ag NPs (per weight) in the chitosan composites provided higher bactericidal and anti-fungal activities.

Antiviral activity of silver nanoparticle/chitosan composites against H1N1 influenza A virus.

Silver nanoparticle (Ag NP)/chitosan (Ch) composites with antiviral activity against H1N1 influenza A virus were prepared. The Ag NP/Ch composites were obtained as yellow or brown floc-like powders following reaction at room temperature in aqueous medium. Ag NPs (3.5, 6.5, and 12.9 nm average diameters) were embedded into the chitosan matrix without aggregation or size alternation. The antiviral activity of the Ag NP/Ch composites was evaluated by comparing the TCID50 ratio of viral suspensions treated with the composites to untreated suspensions. For all sizes of Ag NPs tested, antiviral activity against H1N1 influenza A virus increased as the concentration of Ag NPs increased; chitosan alone exhibited no antiviral activity. Size dependence of the Ag NPs on antiviral activity was also observed: antiviral activity was generally stronger with smaller Ag NPs in the composites. These results indicate that Ag NP/Ch composites interacting with viruses exhibit antiviral activity.

The utility of cerebrospinal fluid for the molecular diagnosis of toxoplasmic encephalitis.

The aim of this study was to assess the efficacy of nested polymerase chain reaction (PCR) and the loop-mediated isothermal amplification (LAMP) assay, which were developed to detect and identify toxoplasma parasites in human cerebrospinal fluid (CSF). Nested PCR was performed using primers generated by Dr. L.D. Sibley to target the 18S rDNA instead of the conventionally used primers which target the B1 gene. We also designed Toxoplasma gondii-specific LAMP primers targeting both genes. In vitro detection sensitivity was evaluated using 10-fold serially diluted genomic DNA purified from RH tachyzoites, and clinical sensitivity and specificity were evaluated using clinical CSF samples from 16 patients with toxoplasmic encephalitis (TE) and from 12 patients with other diseases. The 18S rDNA nested PCR showed the highest detection sensitivity limit with a minimum of 1.0 × 10(-8) ng/µL. However, sensitivity and specificity of nested PCR with clinical specimens were 50% and 100%, respectively. The sensitivity of molecular diagnosis of TE is not sufficient; therefore, patients clinically suspected of having TE should be treated promptly. Our molecular diagnostic tool would restrictively facilitate a definitive diagnosis of TE at an early stage in approximately 50% of patients.

Actively induced antigen-specific CD8+ T cells by epitope-bearing parasite pre-infection but not prime/boost virus vector vaccination could ameliorate the course of Plasmodium yoelii blood-stage infection.

The lack of MHC molecules on red blood cells (RBCs) has led to questions regarding the immunological function of CD8(+) T cells against malarial blood-stage (MBS). However, several recent reports contradicting with this concept have suggested that they play an important role in the course of MBS infection. The present study generated genetically engineered murine malaria, Plasmodium yoelii, which expresses a well-defined Trypanosoma cruzi-derived, H-2K(b)-restricted CD8(+) T cell epitope, ANYNFTLV. Prime/boost vaccination by the use of recombinant adenovirus and recombinant modified vaccinia virus Ankara (MVA), which induced an enhanced number of ANYNFTLV-specific CD8(+) T cells, failed to prevent a pathological outcome to occur upon ANYNFTLV-expressing murine MBS infection. This outcome did not change even with the combination of passive transfer of an appreciable number of in vitro-expanded ANYNFTLV-specific CD8(+) T cells. In contrast, the pre-infection of mice with T. cruzi, which intrinsically bears the same CD8(+) T cell epitope significantly improved the survival of ANYNFTLV-expressing malaria-infected mice but not that of control malaria-infected ones. This protective effect was abrogated by the use of a CD8(+) T cell-depleting monoclonal antibody. Although the protective effect was observed only in certain situations, the actively induced antigen-specific CD8(+) T cells could ameliorate the pathologies caused by the MBS. This is the first study to implicate that the active induction of antigen-specific CD8(+) T cells should be included in the development of a vaccine against MBS.

Quantitative and qualitative features of heterologous virus-vector-induced antigen-specific CD8+ T cells against Trypanosoma cruzi infection.

We studied some aspects of the quantitative and qualitative features of heterologous recombinant (re) virus-vector-induced, antigen-specific CD8(+) T cells against Trypanosoma cruzi. We used three different, highly attenuated re-viruses, i.e., influenza virus, adenovirus and vaccinia virus, which all expressed a single, T. cruzi antigen-derived CD8(+) T-cell epitope. The use of two out of three vectors or the triple virus-vector vaccination regimen not only confirmed that the re-vaccinia virus, which was placed last in order for sequential immunisation, was an effective booster for the CD8(+) T-cell immunity in terms of the number of antigen-specific CD8(+) T cells, but also demonstrated that (i) the majority of cells exhibit the effector memory (T(EM)) phenotype, (ii) robustly secrete IFN-γ, (iii) express higher intensity of the CD122 molecule and (iv) present protective activity against T. cruzi infection. In contrast, placing the re-influenza virus last in sequential immunisation had a detrimental effect on the quantitative and qualitative features of CD8(+) T cells. The triple virus-vector vaccination was more effective at inducing a stronger CD8(+) T-cell immunity than using two re-viruses. The different quantitative and qualitative features of CD8(+) T cells induced by different immunisation regimens support the notion that the refinement of the best choice of multiple virus-vector combinations is indispensable for the induction of a maximum number of CD8(+) T cells of high quality.

CD4(+)CD8(+) thymocytes are induced to cell death by a small dose of puromycin via ER stress.

When the CD4(+)CD8(+) thymic lymphoma cells were treated with puromycin, we found that most of the cells died at 0.3-1 microg/ml of puromycin within 24h. However, cell death was greatly reduced when the dose of puromycin was increased. Similar dose-pattern of cell death was observed in thymocytes and the sensitivity to puromycin was greater in CD4(+)CD8(+) thymocytes than CD4(+)CD8(-) thymocytes. The induction of apoptosis was blocked by the protein synthesis inhibitor cycloheximide, and to some extent by transfection of Bcl-xL or Bcl-2 genes. Expression of GRP78 was up-regulated after treatment with a small dose of puromycin, and the cell death by puromycin was blocked in the presence of caspase 12 inhibitor. These results indicated that the induction of cell death by low-dose puromycin was due to endoplasmic reticulum stress. Furthermore, we found that dexamethasone, a synthetic glucocorticoid, and puromycin worked synergistically to induce cell death in thymocytes.

Miniplates as skeletal anchorage for treating mandibular second molar impactions.

Mandibular second molar impactions can be difficult to correct and might require surgery. A young man with an impacted mandibular right second molar was treated with a miniplate, which provided anchorage to upright the tooth. Although other devices are available, this technique appears to be predictable and quick, and has few side effects.

Trypanosoma cruzi infection from the view of CD8+ T cell immunity--an infection model for developing T cell vaccine.

Chagas' disease is caused by Trypanosoma cruzi (T. cruzi) which was once prevalent in Central and South America. Although the recent success in Triatoma vector control has made the disease being possibly "extinct" in the near future, the development of effective preventive and therapeutic vaccines is still necessary to prevent the resurgence of the neglected infection. In addition to the importance for containing the disease, T. cruzi infection presents unique features for elucidating hosts' immune responses against intracellular infectious agents. Due to its biological capacity for invading into principally any types of cells and for causing systemic infection which damages particularly muscle and neural cells, T cell immunity is critical for resolving its infection. Although T cell-mediated immune responses have been, so far, extensively investigated in viral and bacterial infections, parasitic infection such as malaria has presented epoch-making discovery in T cell immunity. Recent advances in the analyses of T cell-mediated immune responses against T. cruzi infection now make this infectious disease potentially more suitable for detecting subtle immunological changes in hosts' immune defense upon modifying immune system. The current review focuses on the usefulness of T. cruzi infection as a model for developing effective CD8(+) T cell-mediated vaccine against intracellular infectious agents.