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.

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.

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.

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.

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.

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.

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.

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.

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...

Identificación de las especies ecuatorianas de Leishmania por ELISA, utilizando anticuerpos monoclonales

Ciento veinte stocks de Leishmania, aislados de humanos, reservorios y vectores en 40 áreas diferentes de 13 provincias del Ecuador, fueron clasificados en 7 grupos por medio de la técnica de ELISA y 9 anticuerpos monoclonales específicos de especie. Ochenta y nueve de 125 aislamientos fueron identificados como L. (V) panamensis, 15 como L. (L) mexicana, 4 como L (L) amazonensis, 4 como L (V) brazilensis, 3 como L (L) major-like y 3 como L. (V) equatorensis. No se pudo determinar claramente la clasificación de 7 aislamientos.

Variaciones de la secuencia de nucleótidos en el gen I de subunidad de cytocromo C oxidasa, en especies de Leishmania

Se realizó la determinación de las secuencias parciales de nucleótidos de genes COI de 8 especies de Leishmania como: L (Leishmania) amazonensis, L.(Viannia) equatorensis. L.(V) major-like, L. (L) mexicana, L.(V) major, L.(V) guayanensis, L.(V) brazilensis y L.(V) panamensis por el método de PCR, utilizando primers diseñados para genes COI de paramecium. El presente estudio demostró que los primers son útiles para la amplificación de los genes COI de 4 especies: L.(V) major, L(V) guayanensis, L(V) braziliensis y L.(V) panamensis. Se determinó una secuencia parcial de genes COI de las 4 especies.

Un ensayo para detectar parásitos Leishmania en biopsias de piel embebidas en parafina utilizando la reacción en cadena de la polimerasa

Tratamos de detectar amastigotes de Leishmania en 11 biopsias de piel, ordinariamente fijadas en formalina y embebidas en parafina, de pecientes con leishmaniasis cutánea en el Ecuador, por medio de la reacción en cadena de la polimerasa (PCR), utilizando primers específicos de organismos Leishmania que ya han sido reportados. En el presente estudio no fue posible confirmar el ADN amplificado específicamente por PCR, utilizando el ADN gemónico extraído de protozoarios Leishmania. Sin embargo, pudimos detectar solamente el ADN específico en algunos de los especímenes de piel fijados en formalina y embebidos en parafina. Modificando tal técnica, sería necesario establecer métodos más convenientes y confiables para detectar amastigotes de Leishmania por PCR...

Estudios preliminares sobre el diagnóstico de leishmaniasis cutánea utilizando muestras de biopsias de piel por reacción en cadena de polimerasa

Se estudiaron métodos de diagnóstico de leishmaniasis cutánea por reacción en cadena de la polimerasa (PCR), utilizando parásitos vultivados y muestras de biopsias de piel del Ecuador. Se prepararon template ADNs por ebullición por 10 minutos en soluciones Chelex al 5 por ciento. Los parásitos Leishmania fueron detectados por PCR, utilizando primers designados desdel el minicírculo (13A y 13B)y gen mini-exon (S-1629 y S-1630. Los primers primero mencionados amplificaron productos no específicos en ADN humano, y la sensibilidad de la reacción fue baja. Los últimos nunca amplificaron productos específicos aún en templete humano y posibilitaron la identificación a nivel de subgénero. Cuando se aumentó la sensibilidad...

Investigación clínica de la leishmaniasis cutánea en el Ecuador por 5 años (1991-1995)

En este estudio analizamos datos y clínicos y epidemiológicos sobre las alteraciones cutáneas de la leishmaniasis. Un total de 348 pacientes con leishmaniasis cutánea residentes en Ecuador fueron examinados para este estudio. Las áreas de estudio fueron las provincias de Manabí, Los Ríos, Azuay y Esmeraldas. Estas áreas estan localizadas en la costa del pacífico y las estribaciones de los Andes. Cada paciente fue cuidadosamente examinado, clínica y parasitológicamente. De los 348 pacientes en este estudio, 204 fueron varones y 144 mujeres. La edad promedio de los pacientes fue 21.2 años (+-1.2 s.e) en los hombres, 18.9 años (+- 1.4 s.e) son mujeres y 20.2 años (+-0.9 s.e) en total. Los pacientes de menos de 20 años...

Similaridad karyotipo de aislamientos de Leishmania de pacientes, flebotominos, y un perro doméstico, identificando la cepa L mexicana como el agente causal de la leishmaniasis cutánea en los Andes ecuatorianos

Los karyotipos DNA de Leishmania, aislados en 3 comunidades diferentes de los Andes ecuatorianos fueron examinados por electroforesis en gel agarosa de campo pulsado. La similaridad en el karyotipo fue detectada en 12 aislamientos; 4 aislamientos humanos y uno canino, aislados en Paute, 4 aislamientos y 1 de flebotomino aislados en Huigra, y 2 aislamientos de flebotominos en Alausí. Las distancias horizontales entre Paute y Alausí, y entre Alausí y Huigra, son de alrededor de 80 km y 20 km respectivamente. El patrón de banda de el DNA cromosómico de estos aislamientos, se caracterizó por una cadena cromosómica ordenada, especialmente por la presencia de 4 cromosomas de bajo peso molecular de 220, 250 y 325 kilobases...

Anticuerpos monoclonales específicos de especies desarrollados contra Leishmania (Viannia) equatorensis

Se obtuvieron anticuerpos monoclonales contra promastigotes de Leishmania (Viannia), que fue recientemente descrita como una nueva especie (Grimaldi et al., 1992) Ratones BALB/c fueron inmunizados con homogenados de L. (v) equatorensis (promastigotes) obtenidos de un cultivo in vitro de 10 días. La fusión de células esplénicas inmunizadas con células de mieloma P3-X63-Ag8,6.5.3 resultó en la producción de 6 anticuerpos monoclonales (AcMs) contra L. (v) equatorensis. Entre estos, 5, AcMs 9F4, 7H6, 3A7, 8C1 y 1G5 se revelaron como específicos contra L (v) equatorensis por medio de ELISA contra un panel cruzado de cepas de Leishmania. Estos AcMs constituirán, por tanto, un útil incremento para el panel de ancicuerpos...

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...

Evaluación in vitro de la actividad anti-promastigote de Leishmania de lotes de Glucantime (antimoniato de meglumina)

La actividad inhibitoria del crecimiento de promastigotes de Leishmania fue evaluada con 3 diferentes lotes de producción de antimoniato de meglumina (Glucantime), que ha venido siendo usado para el tratamiento de la leishmaniasis en el Ecuador. Por lo menos dos veces se detectaron diferencias en la actividad anti-promastigote de los diferentes lotes de Glucantime. La concentración efectiva de la droga que inhibió la proliferación de los promastigotes en un 50 por ciento (CE50) varió con diferentes especies de Leishmania, y los valores de CE50 de los lotes más efectivos estuvieron en el rango de 20-38 mg/m1 de Glucantine o 5.7-10.8 mg/m1 de antimonio.