Photothermal therapeutic ability of copper open-shell nanostructures that are effective in the second biological transparency window based on symmetry breaking-induced plasmonic properties.

In this study, a photothermal therapy agent that works efficiently in the second biological transparency window was developed based on the localized surface plasmon (LSP) resonance of symmetry-broken open-shell nanostructures of low-cost Cu (CuOSNs). The strong LSP resonance and superior photothermal conversion ability in the second biological transparency window were achieved by generating the dipolar bonding mode due to the plasmon hybridization between the nanoshell dipole and the nanohole dipole at the opening edge in CuOSNs derived from the symmetry breaking of a Cu nanoshell. Oxidative dissolution of CuOSNs in water was significantly suppressed by successive coating with the self-assembled monolayer of 16-mercaptohexadecanoic acid and a thin silica layer. Furthermore, the stability in phosphate buffered saline, which models the biological environment, was attained by further coating the nanoparticles with polyethylene glycol. It was demonstrated from in vitro cell tests using HeLa cells that the cytotoxicity of CuOSNs was effectively suppressed by the surface protection. The viability of HeLa cells incubated with CuOSNs was decreased under the irradiation of low intensity 1060 nm laser with increasing number of CuOSNs. These results demonstrate that low-cost symmetry-broken Cu-based nanostructures can act as an excellent photothermal therapy agent in the second biological transparency window.

Modulation Technique of Localized Surface Plasmon Resonance of Palladium Nanospheres by Coating with Titanium Dioxide Shell for Application to Photothermal Therapy Agent.

Although plasmonic palladium (Pd) nanospheres are thermodynamically stable and have high photothermal conversion due to the free and bound electron coupling associated with the intrinsic high interband transition, they have not attracted attention as a photothermal conversion material for next-generation photothermal cancer therapy. This is because the Pd nanospheres generate the localized surface plasmon resonance (LSPR) intrinsically in the ultraviolet region, which is far away from the biological transparent window (750-900 nm). In this study, we controlled the LSP wavelength of Pd nanospheres by coating with high refractive index TiO2 shells taking advantage of the Pd LSPR which is highly sensitive to changes in the local refractive index around the nanospheres. Our calculations indicated that the absorption cross section at 808 nm (corresponding to the wavelength used for photothermal treatment) was increased by 4.5 times by redshifting the LSPR and increasing the extinction intensity associated with the coating with TiO2 shell. Experiments confirmed the theoretical prediction in that the LSPR of the synthesized Pd nanospheres with a diameter of 81 nm was significantly redshifted by coating with amorphous TiO2 shell, resulting in significant large extinction intensity at 808 nm. The photothermal conversion efficiency was estimated to be 50%. In vitro cell tests, HeLa cells incubated with 100-300 µg/mL TiO2-coated Pd nanospheres were efficiently killed by irradiating 808 nm laser (1.8 W) even though the nanospheres with the same concentrations showed little cytotoxicity. These results indicate that the Pd nanospheres coated with high refractive index shells can be promising as a photothermal therapy agent.

Serological evidence for hepatitis e virus infection in laboratory monkeys and pigs in animal facilities in Japan.

In laboratory animal facilities, monkeys and pigs are used for animal experiments, but the details of hepatitis E virus (HEV) infection in these animals are unknown. The risk of infection from laboratory animals to humans has become a concern; therefore, much attention should be paid to the handling of these animals during their care and use, including surgical procedures performed on infected animals. In this connection, serum samples collected from 916 monkeys and 77 pigs kept in 23 animal facilities belonging to the Japanese Association of Laboratory Animal Facilities of National University Corporations (JALAN) and the Japanese Association of Laboratory Animal Facilities of Public and Private Universities (JALAP) in Japan were examined for the purpose of detecting antibodies to HEV and HEV RNA by using ELISA and RT-PCR, respectively. One hundred and seven serum samples of 916 (11.7%) monkeys were positive for anti-HEV IgG, and 7 and 17 serum samples of 916 (0.8% and 5.3%) monkeys were positive for anti-HEV IgM and IgA, respectively. Thirty-six samples from 62 (58.1%) farm pigs were positive for anti-HEV IgG, whereas all samples tested from miniature pigs were negative (0/15, 0%). Seven samples from 62 (9.1%) farm pigs and 7 samples from 916 (0.8%) monkeys were positive for IgM antibody, but these HEV-IgM antibody positive serum samples were HEV-RNA negative by RT-PCR. The IgM antibody positive rate (9.1%) of farm pigs was much higher than that of monkeys (0.8%). These results suggest the relative levels of risk of HEV infection from these animals to animal handlers and researchers who work with them in laboratory animal facilities.

Characterization of microglia induced from mouse embryonic stem cells and their migration into the brain parenchyma.

We derived microglia from mouse embryonic stem cells (ES cells) at very high density. Using the markers Mac1(+)/CD45(low) and Mac1(+)/CD45(high) to define microglia and macrophages, respectively, we show that Mac1(+) cells are induced by GM-CSF stimulation following neuronal differentiation of mouse ES cells using a five-step method. CD45(low) expression was high and CD45(high) expression was low on induced cells. We used a density gradient method to obtain a large amount of microglia-like cells, approximately 90% of Mac1(+) cells. Microglia-like cells expressed MHC class I, class II, CD40, CD80, CD86, and IFN-gammaR. The expression level of these molecules on microglia-like cells was barely enhanced by IFN-gamma. Intravenously transferred GFP(+) microglia derived from GFP(+) ES cells selectively accumulated in brain but not in peripheral tissues such as spleen and lymph node. GFP(+) cells were detected mainly in corpus callosum and hippocampus but were rarely seen in cerebral cortex, where Iba1, another marker of microglia, is primarily expressed. Furthermore, both GFP(+) and Iba1(+) cells exhibited a ramified morphology characteristic of mature microglia. These studies suggest that ES cell-derived microglia-like cells obtained using our protocol are functional and migrate selectively into the brain but not into peripheral tissues after intravenous transplantation.

Species assignation of Leishmania from human and canine American tegumentary leishmaniasis cases by multilocus enzyme electrophoresis in North Argentina.

Sixteen Leishmania stocks, 15 isolated from patients with cutaneous (CL), mucocutaneous (MCL), or recurrent cutaneous leishmaniasis, plus one from a dog with CL in Salta and Corrientes Provinces, Argentina, were studied by multilocus enzyme electrophoresis. Thirteen of the stocks from humans were grouped in two zymodemes; nine termed as KMS 1, four as KMS 2, and assigned to Leishmania (Viannia) braziliensis. Two additional stocks from CL cases expressed a KMS 4 enzyme profile, corresponding to L. (V.) guyanensis. Although the parasites from the dog were also assigned to L. (V.) braziliensis, its zymodeme, KMS 3, was not expressed in any of the current human isolates. The characterization of Leishmania from a dog was done for the first time in Argentina. The importance of the intraspecific polymorphism in the induction of clinical forms and in the host-reservoir concept is briefly discussed, based on the zymodeme data of isolates from humans and dogs. The presence of L. (V.) guyanensis was confirmed in the country.

IL-5-Induced Eosinophils Suppress the Growth of Leishmania amazonensis In Vivo and Kill Promastigotes In Vitro in Response to Either IL-4 or IFN-gamma.

In IL-5 transgenic mice (C3H/HeN-TgN(IL-5)-Imeg), in which 50% of peripheral blood leukocytes are eosinophils, the development of infection by Leishmania amazonensis was clearly suppressed. To determine mechanistically how this protozoan parasite is killed, we performed in vitro killing experiments. Either IL-4 or IFN-gamma effectively stimulated eosinophils to kill Leishmania amazonensis promastigotes, and most of the killing was inhibited by catalase but not by the NO inhibitor L-N5-(1-iminoethyl)-ornithine, suggesting that hydrogen peroxide is responsible for the killing of L. amazonensis by eosinophils. There was no significant degranulation of eosinophils in the culture, because eosinophil peroxidase was not detected in culture supernatants when L. amazonensis promastigotes were killed by activated eosinophils. Such resistance was also observed in BALB/c mice, which are highly susceptible to L. amazonensis. Expression plasmids for IL-4, IL-5, and IFN-gamma were transferred into muscle by electroporation in vivo starting 1 week before infection. Expression plasmid for IL-5 was most effective in slowing the development of infection among three expression plasmids. Expression plasmid for IL-4 was slightly effective and that for IFN-gamma had no effect on the progress of disease. These results suggest that IL-5 gene transfer into muscle by electroporation is useful as a supplementary protection method against L. amazonensis infection.

Identification of endotrypanum species from a sloth, a squirrel and Lutzomyia sandflies in ecuador by PCR amplification and sequencing of the mini-exon gene.

PCR amplification and nucleotide sequencing of the mini-exon gene revealed that four strains isolated from a sloth (Choloepus hoffmanni), a squirrel (Sciurus granatensis) and two sandflies (Lutzomyia hartmanni) in Ecuador were indistinguishable from Endotrypanum monterogeii. Another strain isolated from Lu. hartmanni showed the high sequence similarity to E. schaudinni. Since three of these strains have been previously identified as Leishmania (Viannia) equatorensis, the results demonstrate that L. (V.) equatorensis is genetically closely related to the genus Endotrypanum. The present study also indicates that Endotrypanum species are distributed in arboreal animals and sandflies in Ecuador, and that mini-exon gene amplification is useful for epidemiological studies of Leishmania and Endotrypanum in the New World.

Development and functional analysis of eosinophils from murine embryonic stem cells.

We have established a culture system for the development of eosinophils from murine embryonic stem (ES) cells. After transferring ES cells from embryonic fibroblast cells onto macrophage colony-stimulating factor-deficient stromal cells, OP9, ES cells were cultured in the presence of interleukin (IL)-5 with either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF) for 20 d to obtain approximately 50% eosinophils. Electron microscopy confirmed the presence of crystallized major basic protein (MBP) in the granules of some of these cells. Neither IL-5, IL-3, GM-CSF nor eotaxin alone could induce eosinophils as efficiently as the conditions described above. Eotaxin induced eosinophil development in combination with either IL-3 or IL-5. Levels of GATA-1, Friend of GATA (FOG)-1, PU.1, CCAAT/enhancer binding protein (C/EBP)alpha, C/EBPbeta, IL-3 receptor alpha (IL-3Ralpha), GM-CSF receptor alpha (GM-CSFRalpha), and MBP mRNAs were increased in ES cells 10 d after transfer onto OP9 cells. In contrast, C/EBPepsilon, IL-5Ralpha, and eosinophil peroxidase mRNAs were induced in response to IL-3 and IL-5 after transfer onto OP9 cells. Eosinophils that developed in this system expressed Gr-1, F4/80, B220, CCR3, IL-3Ralpha, IL-5Ralpha, and DX5. Finally, eosinophils developed from ES cells produced reactive oxygen species in response to Leishmania as do peripheral blood eosinophils.

Mitochondrial transmembrane potential is diminished in phorbol myristate acetate-stimulated peritoneal resident macrophages isolated from wild-type mice, but not in those from gp91-phox-deficient mice.

Macrophages produce superoxide (O2-) during phagocytosis or upon stimulation with a variety of agents including phorbol myristate acetate (PMA) through the activation of NADPH oxidase, and the formed O2- is converted to other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). The aim of the present study was to elucidate the effect of the intracellularly produced ROS on mitochondrial transmembrane potential (MTP) in mouse (C57BL/6) peritoneal resident macrophages stimulated with PMA. Using a fluorescent dye, succinimidyl ester of dichlorodihydrofluorescein (H2DCFDA), O2- was visualized in intracellular compartments in a certain subpopulation of macrophages isolated from wild-type mice. Cells deficient in gp91-phox, one of the membrane components of NADPH oxidase, were negative for the fluorescence. When cells were loaded with both H2DCFDA and MitoCapture, a fluorescent dye for mitochondria, mitochondrial fluorescence was diminished in O2- -producing cells, but not in O2- -deficient cells. Flow cytometry also revealed the decrease of mitochondrial fluorescence in wild-type cells, but not in gp91-phox-deficient cells. The loss of mitochondrial fluorescence was prevented by microinjection of catalase into cells. The present findings demonstrate that MTP is diminished by ROS, including the H2O2 dismutated from O2-, produced intracellularly by activation of the NADPH oxidase in mouse peritoneal resident macrophages stimulated with PMA.

Diagnosis of visceral leishmaniasis by enzyme-linked immunosorbent assay using urine samples.

A diagnostic method has been developed to detect anti-Leishmania donovani immunoglobulin G (IgG) in urine by enzyme-linked immunosorbent assay (ELISA). In measuring anti-L. donovani IgG, IgA, and IgM in urine, the method performed best in the detection of IgG. The sensitivity and specificity of the assay were determined with panels of urine samples from 62 visceral leishmaniasis (VL) patients, 59 healthy controls from areas of endemicity, 53 healthy controls from areas of nonendemicity, 59 malaria patients, 13 tuberculosis patients, 23 cutaneous leishmaniasis patients, and 7 patients with other diseases. Using L. donovani promastigote crude antigen, the test had 93.5% sensitivity (58 positives of 62 VL patient samples) and 89.3% specificity (191 negatives of 214 non-VL patient samples). The ELISA with acetone-treated L. donovani promastigote antigen raised the sensitivity and specificity to 95.0 and 95.3%, respectively. Western blot analysis revealed that most of the samples that cross-reacted with crude antigen in ELISA did not recognize any antigenic component of L. donovani crude antigen. We also checked 40 serum samples from the same group of VL patients for anti-L. donovani IgG and got 90.0% sensitivity with both crude and acetone-treated antigens. As collection of urine is much easier than collection of serum, the detection of anti-L. donovani IgG in urine with acetone-treated antigen will be useful in epidemiological studies. It could be an adjunct of laboratory diagnosis.

Reacción intradérmica (skin test) utilizando antígeno de promastigotes de Leishmania, en individuos de áreas endémicas de la costa y sierra del Ecuador

Un total de 229 pacientes individuos de las áreas endémicas de la costa y sierra del Ecuador, fueron examinados realizando skin test y observaciones dermatológicas. No se encontraron diferencias importantes en el grado de positividad del skin test entre individuos de las dos regiones. Sin embargo, una reacción fuertemente marcada se observó en sujetos de las alturas. Este tipo de reacción podría ser causado por la diferencia de la respuesta inmune de sujetos provenientes de las dos áreas endémicas del Ecuador.

Respuestas de pruebas intradérmicas con antígeno parcialmente purificado obtenido de promastigotes de leishmania panamensis en la leishmaniasis cutánea activa

El presente estudio fue diseñado para evaluar los antígenos preparados a partir de promastigotes de Leishmania panamensis para evaluar el skin test en pacientes con leishmaniasis cutánea. El antígeno crudo preparado (CA) utilizado el sobredadante del centrifugado de homogenado de parásito a 10,000 xg. El extracto soluble fue luego sometido a 4 preparaciones, denominadas FA-1 a Fa-4, con la ayuda de un Sephacryl S-200 de filtración en gel. Los parásitos enteros (5x10 parásitos por examen) preparados en salina fenolizada, fueron utilizados como un antígeno para Montenegro (MA). El skin test intradérmico fue hecho en 17 pacientes con leishmaniasis cutánea activa causada por Le. braziliensis (complejo). El radio positivo...

Análisis del karyotipo de Leishmania (Viannia) panamensis aislada de pacientes con leishmaniasis cutánea de la costa del pacífico en el Ecuador

El cariotipo ADN de aislamientos de Leishmania de pacientes con la forma cutánea de la enfermedad procedente de 9 zonas endémicas de la costa del pacífico del Ecuador, fueron analizados por electroforesis en gel de campo pulsado. Todos los stocks aislados, incluyendo aquellos de una misma área endémica, presentaron un patrón de bandas de ADN cromosómico diferente. La variación de tamaño en las bandas de ADN cromosómico resultó evidentemente particular en los cromosomas entre 400 y 800 kb. Sin embargo, se observaron también bandas de ADN de aproximadamente 230, 350, 800 y 850 kb. Sin embargo, se observaron también bandas de ADN de aproximadamente 230, 350, 800 y 850 kb. Debido a que un cariotipo ADN semejante ha sido reportado...

Evaluación de la intradermo reacción y ELISA en el estudio epidemiológico de la leishmaniasis

El presente estudio fue diseñado para evaluar individual y comparativamente, la intradermo reacción y ELISA, como elementos de diagnóstico en el muestreo de la leishmaniasis cutánea y mucocutánea del Ecuador. El antígeno para la intradermo reacción fue preparado fragmentando promastigotes de L. braziliensis. Un estudio preliminar de intradermo reacción utilizando este antígeno, fue desarrollado en 63 individuos con lesiones dérmicas activas, junto a la prueba de ELISA. Los grados de positividad de ambas pruebas fueron significativamente altos en aquellos individuos cuyas lesiones fueron positivas para el parásito, demostrando alta sensibilidad y especificidad en los pacientes con leishmaniasis. Un estudio epidemiológico...

Examen de los hospedadores reservorios de la leishmaniasis usando contra-inmunoelectroforesis

Este examen fue diseñado para detectar antígenos de leishmania en los tejidos de mamíferos silvestres capturados en las áreas endémicas de leishmaniasis en el Ecuador. La detección de los antígenos fue realizada por contra-inmunoelectroforesis (CIE). Las líneas de precipitina fueron observadas entre el suero anti-l.b brazilensis y extracto de hígado (antígeno) obtenido de 3 Didelphis marsupialis, 2 el antisuero, y un extracto de bazo de c. lanatus, que ya había presentado CIE positiva para extracto de reservorios de la leishmaniasis en el Ecuador, para D. marsupialis y C. lanatus, tal incriminación fue hecha por primera vez.

Leishmania aislada de humanos en Ecuador

Durante el presente estudio, se obtuvieron 18 aislamientos del parásito Leishmania de pacientes de la costa del Pacífico y región amazónica, y 11 de la sierra o región interandina, todos a partir de lesiones ulcerosas. La distribución geográfica y los aspectos clínico y parasitológico son brevemente considerados. Parece que es más factible lograr aislamientos del parásito en la leishmaniasis de las tierras bajas en las primeras semanas de evolución, aunque es relativamente fácil lograrlo también en etapas tardías en la región andina o tierras altas.

Un nuevo ensayo de aislamiento de leishmania de animales domésticos y silvestres en Ecuador

A fin de identificar los hospedadores reservorios animales en diferentes áreas endémicas de leishmaniasis humana, 194 animales domésticos y silvestres fueron examinados en busca de parásitos. Por el método de punción hepática, 14 (7,2 por ciento) del total, resultaron positivos con protozoarios. Una cepa de perro doméstico de los Andes fue identificada como L. pifanoi (más tarde identificada como Le. mexicana) (Tesh y Grimaldi, comunicación personal). Otras sin embargo, permanecen inidentificadas, en busca de un método preciso de caracterización, utilizando electroforesis de isoenzimas (Kreutzer, comunicación personal).

Análisis cariotipo de aislamientos de leishmania del Ecuador por electroforesis de campo pulsado

Los cariotipos de los parásitos Leishmania mexicana, L. panamensis y L. major-like recientemente aislados en Ecuador fueron analizados por medio de electroforesis en gel de campo pulsado (PFGE). Un total de 18-21 cromosomas de 200 kb a más de 1,100 kb fueron resueltos, dependiendo de los aislamientos, utilizando un aparato graduable de electroforesis en gel de campo pulsado. El PFGE reveló cariotipos de DNA específicos de las especies para tres diferentes tipos de Leishmania. El cariotipo de L. mexicana fue casi idéntico entre cuatro aislamientos incluyendo tres de pacientes y uno por un perro en una pequeña comunidad andina, sugiriendo así que los perros domésticos pueden desempeñar un papel como hospedadores reservorios...

Observaciones histológicas de hámsters dorados infectados con una cepa ecuatoriana de Leishmania mexiacana

Se desarrolló un estudio experimental para investigar el efecto de la infección de hámsters dorados con Leishmania mexicana. Los animales fueron infectados con L. mexicana de Ecuador. En la autopsia, seis meses después de la inoculación, los sitios de infección se habían tornado en lesiones ulcerativas exudativas inflamatorias cubiertas con gruesas costras. No se observó metástasis cutáneas en otras partes expuestas del cuerpo. Histológicamente las muestras de la nariz y las plantas de los pies mortraban gran número de amastigotes, con infiltración extensiva de histiocitos, linfocitos, y alguna cantidad de neutrófilos, eosinófilos y células plasmáticas. Un importante número de células mastoideas era predominante...

Comparación histopatológica y estructural de leishmaniasis animal experimental causada por diferentes cepas de Leishmania (Leishmania) mexicana aislada de pacientes con lesiones cutáneas localizadas y difusas

Con la finalidad de investigar algunos factores relacionados a diferentes formas clínicas, causadas por cepas o especies de Leishmania, se realizaron comparaciones histopatológicas y ultraestructurales. Con este propósito, se infectaron hámsters con promastigotes de Leishmania (Leishmania) mexicana, aislados de pacientes con dos tipos de formas clínicas, leishmaniasis cutáneo-difusa (LCD) y leishmaniasis cutánea lozalizada (LCL). No se encontraron aspectos histopatológicos ni ultraestructurales que indicasen un clara diferenciación entre las cepas de LCD y LCL. Los animales de experimentación utilizados fueron dividos en dos grupos como sigue: los hámsters en el grupo A fueron infectados con L (L) mexicana, aislada...