Killing Bacteria by Faradaic Processes through Nano-Hydroxyapatite/MoOx Platforms.

Following the secular idea of ″restitutio ad integrum″, regeneration is the pursued option to restore bones lost after a disease; accordingly, complementing antibiotic and regeneration capacity to bone grafts represents a great scientific success. This study is a framework proposal for understanding the antimicrobial effect of biocompatible nano-hydroxyapatite/MoOx (nano-HA/MoOx) platforms on the basis of their electroactive behavior. Through cyclic voltammetry and chronoamperometry measurements, the electron transference capacity of nano-HA and nano-HA/MoOx electrodes was determined in the presence of pathogenic organisms: Pseudomonas aeruginosa and Staphylococcus aureus. Faradaic processes were confirmed and related to the switch of MoO42-/PO43- groups in the original hexagonal nano-HA crystal lattice and to the extent of OH vacancies that act as electron acceptors. Microscopic analysis of bacteria's ultrastructure showed a disruptive effect on the cytoplasmic membrane upon direct contact with the materials, which is not evident in the presence of eukaryotic cells. Experiments support the existence of a type of extracellular electron transfer (EET) process that alters the function of the bacterial cytoplasmic membrane, accelerating their death. Our findings provide strong quantitative support for a drug-independent biocidal physical approach based on EET processes between microorganisms and phosphate ceramics that can be used to combat local orthopedic infections associated with implants.

Development of a New LAMP Assay for the Detection of Ancylostoma caninum DNA (Copro-LAMPAc) in Dog Fecal Samples.

Ancylostoma caninum is a zoonotic nematode which is able to affect animals and humans. Diagnosis in the definitive host and environmental detection are key to prevent its dissemination and achieve control. Herein, a new coprological LAMP method for the detection of A. caninum (Copro-LAMPAc) DNA was developed. DNA extraction was performed using a low-cost method and a fragment of the cox-1 gene was used for primer design. The analytical sensitivity, evaluated with serial dilutions of genomic DNA from A. caninum adult worms, was 100 fg. A specificity of 100% was obtained using genomic DNA from the host and other pathogens. The Copro-LAMPAc was evaluated using environmental canine fecal samples. When compared with gold standard optical microscopy in epidemiological studies, it proved to be more sensitive. This new LAMP assay can provide an alternative protocol for screening and identification of A. caninum for epidemiological studies in endemic areas.

Circulación del parásito Echinococcus granulosus en Los Antiguos, Santa Cruz / Circulation of Echinococcus granulosus in Los Antiguos, Santa Cruz

Resumen La hidatidosis, causada por el parásito Echinococcus granulosus, es una zoonosis endémica en la provincia de Santa Cruz asociada a áreas de producción ganadera. El hombre puede permanecer asintomático durante un largo período luego de la infección. Una vez desarrollada, la enfermedad representa un importante problema de salud pública debido a la complejidad y el costo de su tratamiento. Los objetivos del trabajo fueron determinar la prevalencia de contaminación ambiental por E. granulosus en zona rural y periurbana de la localidad de Los Antiguos, mediante la detección de antígenos específicos en heces caninas, e identificar factores de riesgo de transmisión. Entre mayo-2016 y abril-2017, se visitaron 38 chacras periurbanas y estancias rurales, definidas como "unidades epidemiológicas". Se analizaron 144 muestras de heces de caninos con téc nica copro-ELISA. Se realizó una encuesta epidemiológica de los pobladores rurales sobre patrones culturales relacionados a la hidatidosis. Se encontró un índice de contaminación ambiental del 17.3% y el 44.7% de las unidades epidemiológicas fueron positivas. Se identificaron prácticas de riesgo como faena domiciliaria (34.2%), alimentación de caninos con vísceras crudas (52.6%), y ausencia de desparasitación de perros (86.8%). Alrededor de la mitad de la población encuestada desconocía las formas de contagio y las medidas de prevención de la enfermedad. Este trabajo muestra un índice alto de contaminación y establece una línea de base para realizar comparaciones a futuro. También refuerza la necesidad de implementar medidas de educación, prevención y control de hidatidosis a nivel local de acuerdo a los programas nacionales.

Abstract Hydatidosis − caused by the parasite Echinococcus granulosus − is a zoonosis endemic to the province of Santa Cruz, associated with areas of livestock production. Once infected, man may remain asymptomatic for a prolonged pe riod but the disease has an important impact on public health owing to the complexity and costs of its treatment. This study aimed at assessing the prevalence of environmental E. granulosus infection in rural and peri-urban areas of the town of Los Antiguos, through the detection of antigens in scattered canine feces, and to identify risk factors for transmission. From May-2016 to April-2017, 38 peri-urban and rural farms, defined as "epidemio logical units", were visited; 144 samples of canine feces were analyzed with the copro-ELISA technique. Rural settlers were enrolled in an epidemiological survey on cultural patterns related to hydatidosis. An environmental contamination index of 17.3% was found and 44.7% of the epidemiological units were positive. Risk practices were found, such as domiciliary slaughter (34.2%), canine feeding with raw viscera (52.6%), and lack of dog de worming (86.8%). In turn, about half of the surveyed population ignored the modes of transmission of the infection and the measures to prevent it. This work shows a high infection index in the area and establishes a baseline for future comparisons. It also reinforces the need to implement education, prevention, and control activities at the local level − according to national program guidelines − in order to reduce the prevalence of environmental contamination of the disease.

[Circulation of Echinococcus granulosus in Los Antiguos, Santa Cruz]. / Circulación del parásito Echinococcus granulosus en Los Antiguos, Santa Cruz.

Hydatidosis - caused by the parasite Echinococcus granulosus - is a zoonosis endemic to the province of Santa Cruz, associated with areas of livestock production. Once infected, man may remain asymptomatic for a prolonged period but the disease has an important impact on public health owing to the complexity and costs of its treatment. This study aimed at assessing the prevalence of environmental E. granulosus infection in rural and peri-urban areas of the town of Los Antiguos, through the detection of antigens in scattered canine feces, and to identify risk factors for transmission. From May-2016 to April-2017, 38 peri-urban and rural farms, defined as "epidemiological units", were visited; 144 samples of canine feces were analyzed with the copro-ELISA technique. Rural settlers were enrolled in an epidemiological survey on cultural patterns related to hydatidosis. An environmental contamination index of 17.3% was found and 44.7% of the epidemiological units were positive. Risk practices were found, such as domiciliary slaughter (34.2%), canine feeding with raw viscera (52.6%), and lack of dog deworming (86.8%). In turn, about half of the surveyed population ignored the modes of transmission of the infection and the measures to prevent it. This work shows a high infection index in the area and establishes a baseline for future comparisons. It also reinforces the need to implement education, prevention, and control activities at the local level - according to national program guidelines - in order to reduce the prevalence of environmental contamination of the disease.

La hidatidosis, causada por el parásito Echinococcus granulosus, es una zoonosis endémica en la provincia de Santa Cruz asociada a áreas de producción ganadera. El hombre puede permanecer asintomático durante un largo período luego de la infección. Una vez desarrollada, la enfermedad representa un importante problema de salud pública debido a la complejidad y el costo de su tratamiento. Los objetivos del trabajo fueron determinar la prevalencia de contaminación ambiental por E. granulosus en zona rural y periurbana de la localidad de Los Antiguos, mediante la detección de antígenos específicos en heces caninas, e identificar factores de riesgo de transmisión. Entre mayo-2016 y abril-2017, se visitaron 38 chacras periurbanas y estancias rurales, definidas como "unidades epidemiológicas". Se analizaron 144 muestras de heces de caninos con técnica copro-ELISA. Se realizó una encuesta epidemiológica de los pobladores rurales sobre patrones culturales relacionados a la hidatidosis. Se encontró un índice de contaminación ambiental del 17.3% y el 44.7% de las unidades epidemiológicas fueron positivas. Se identificaron prácticas de riesgo como faena domiciliaria (34.2%), alimentación de caninos con vísceras crudas (52.6%), y ausencia de desparasitación de perros (86.8%). Alrededor de la mitad de la población encuestada desconocía las formas de contagio y las medidas de prevención de la enfermedad. Este trabajo muestra un índice alto de contaminación y establece una línea de base para realizar comparaciones a futuro. También refuerza la necesidad de implementar medidas de educación, prevención y control de hidatidosis a nivel local de acuerdo a los programas nacionales.

Humoral immune response of pigs infected with Toxocara cati.

Toxocara cati is one of the causative agents of human toxocariasis. Serological methods are used for diagnosis in paratenic hosts like humans but the humoral immune response triggered by this parasite is unknown. We characterized the humoral immune response to T. cati excretory-secretory antigens (TES) in pigs as animal model during the acute and chronic stages of infection. ELISA and Western Blot techniques were used to determine antibody response. Pigs were experimentally inoculated with 100,000 infective Toxocara cati eggs. Blood was collected at 7, 14, 21 and 28 days post-inoculation (d.p.i.) to assess the acute stage of infection and 90, 120 and 180 d. p.i. for chronic stage analysis. ELISA showed values higher than the cut-off of specific IgM and IgG at 7 d. p.i. with significant differences at 0 and 7 d. p.i. for IgM and at 14, 21 and 28 d. p.i. for IgG in the acute stage. Higher and stable levels were detected in the chronic stage. Western Blot showed bands from 102 to 38 kDa detected by specific IgM and IgG. More immunogenic bands were identified by specific IgG. In the chronic stage of infection a band near 31 kDa was the only band detected by IgM until 150 d. p.i. Specific IgG recognized bands between 102 and 31 kDa. This study demonstrates how the humoral immune response evolves in the acute and chronic stages of infection and provides evidence on the role of the pig as a paratenic host of T. cati.

Antibacterial alginate/nano-hydroxyapatite composites for bone tissue engineering: Assessment of their bioactivity, biocompatibility, and antibacterial activity.

Bone substitute materials based on bioceramics and polymers have evolved shifting from a passive role where they are merely accepted by the body; to an active role, where they respond to particular environmental conditions or to different types of cues generating suitable integration (osseointegration for this case) inside the host tissue. In this work, two types of composite materials based on a bioceramic (synthetic nano-hydroxyapatite, HA) and a biopolymer (sodium alginate, ALG) have been designed and assessed for promoting the bone regeneration. These materials were loaded with ciprofloxacin (CIP) for obtaining, not only a suitable material for a filling but with antibacterial properties. Therefore, their main features were studied through Fourier transformed-infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and transmission electron microscopy (TEM). Ultraviolet-Visible (UV-Vis) spectroscopy was used for obtaining the released concentrations of CIP and Zeta-potential (ζ-potential) was used for characterizing the adsorption of CIP onto nanoparticles. The release profile of this drug has been fit with the Ritger-Peppas model, used for studying the release kinetics of hydrogel-based systems. The bioactivity of these composites was also evaluated after 30 days of incubation in a simulated body fluid solution (SBF). Then, the assessment of antibacterial capability against the three main strains cause osteomyelitis was performed. Finally, the cell viability study and the cellular morphology assay were also carried out. These last assays have shown encouraging results and, gathered with their other properties, such as their bioactivity and antibacterial properties; they could lead to propose these materials as new bone filler antibiotic devices.

Identification of potential 'hot spots' of cystic echinococcosis transmission in the province of Río Negro, Argentina.

Cystic echinococcosis (CE) is a parasitic zoonosis caused by Echinococcus granulosus. The control program of CE of Rio Negro province, Argentina, involves annual surveillance using ultrasound (US) screening in school children, and five-year cross-sectional surveys to detect livestock farms with parasitized dogs by coproELISA with confirmation tests (Western Blot or PCR). Control program is based on deworming of dogs with praziquantel and the aim is to identify areas at risk of Cystic echinococcosis transmission to humans, using all available data sources. The information was spatially distributed in 13 program areas and, at a smaller geographical scale, in 80 Primary Health Care Centers. CoproELISA surveys involved three randomized sampling periods (2003-05, 2009-10, 2017-18), with 1790 canine fecal samples. The US surveys were conducted in 2003-08, 2009-16 and 2017-18 in 34,515 children. Heat maps were created at the smallest geographic scale with QGIS 3.4.6. For the consecutive sampling periods, prevalence of positive canine fecal samples from livestock farms were 14.7, 12.1 and 7.8%, respectively, and children prevalence was 0.4, 0.2 and 0.1%, respectively. The study has been developed on a scale according to which the temporal-spatial distribution of CE allows to adjust control strategies in those areas of potential transmission of the zoonosis to humans.

First inter-laboratory comparison of Echinococcus granulosus sensu lato diagnosis in Latin America

[ABSTRACT]. Objective. To compare the performance of polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) tests for diagnosing Echinococcus granulosus in dog feces among national reference laboratories in Argentina, Chile, Peru, and Uruguay. Methods. National laboratories affiliated with the Ministry of Health/Agriculture of each country exchanged panels of 10 positive/negative samples obtained from their regular national surveillance programs in November 2015 – November 2016. All laboratories applied PCR; two also applied ELISA techniques. Sensitivity and specificity were determined for each laboratory and concordance of results among the laboratories was evaluated by Cohen Kappa coefficient. Results. Poor concordance (3 of 10 paired comparisons had values of Kappa > 0.4), low sensitivity and specificity across all laboratories, and poor performance of both techniques in detecting E. granulosus in canine feces was demonstrated in this study. An ex-post comparison of the laboratories’ test protocols showed substantial heterogeneity that could partially explain poor concordance of results. Conclusion. The results underscore the heterogeneity of canine echinococcosis diagnosis across the region and indicate possible sources of variability. Efforts to standardize canine echinococcosis testing must be included in the plan of action for the Regional Initiative for the Control of Cystic Echinococcosis. Future comparisons with fecal samples of known parasite load are needed.

[RESUMEN]. Objetivo. Comparar el rendimiento de los ensayos de la reacción en cadena de la polimerasa y el enzimoinmunoanálisis de adsorción en fase sólida (o ELISA, por su sigla en inglés) para diagnosticar Echinococcus granulosus en heces caninas en los laboratorios de referencia nacionales de Argentina, Chile, Perú y Uruguay. Métodos. Los laboratorios nacionales, afiliados a los ministerios de salud y agricultura y ganadería de cada país, intercambiaron paneles de diez muestras positivas y negativas obtenidas de sus respectivos programas nacionales de vigilancia desde el mes de noviembre del año 2015 hasta el mismo mes del año siguiente. Todos los laboratorios emplearon la reacción en cadena de la polimerasa y dos emplearon también técnicas de ensayo inmunoenzimático (ELISA). Se determinó la sensibilidad y la especificidad de cada laboratorio y se evaluó la concordancia entre los resultados de los laboratorios mediante el coeficiente kappa de Cohen. Resultados. Este estudio descubrió una escasa concordancia (3 de 10 comparaciones de pares obtuvieron valores de kappa > 0,4), una sensibilidad y especificidad bajas en todos los laboratorios y un rendimiento deficiente de ambas técnicas de diagnóstico de Echinococcus granulosus en heces caninas. La comparación ex post de los protocolos de ensayo de los laboratorios mostró una heterogeneidad sustancial que podría explicar parcialmente la escasa concordancia de los resultados. Conclusiones. Los resultados subrayan la heterogeneidad del diagnóstico de equinococosis canina en toda la región e indican posibles fuentes de esta variabilidad. Deben incluirse medidas para estandarizar la prueba de equinococosis canina en el plan de acción de la Iniciativa Sudamericana para el Control de la Equinococosis Quística. En el futuro serán necesarias comparaciones adicionales con muestras fecales con una carga de parásitos conocida.

[RESUMO]. Objetivo. Comparar o desempenho dos métodos de reação em cadeia da polimerase (PCR) e ensaio imunoenzimático (ELISA) no diagnóstico de infecção pelo Echinococcus granulosus em fezes de cães entre laboratórios de referência nacional na Argentina, Chile, Peru e Uruguai. Métodos. Laboratórios nacionais conveniados ao Ministério da Saúde/Agricultura de cada país participante intercambiaram grupos de 10 amostras positivas/negativas coletadas rotineiramente pelos programas nacionais de vigilância no período de novembro de 2015 a novembro de 2016. Todos os laboratórios empregaram o método de PCR e dois empregaram também o método de ELISA. A sensibilidade e a especificidade dos métodos foram determinadas em cada laboratório, e a concordância dos resultados entre os laboratórios participantes foi avaliada com o coeficiente kappa de Cohen. Resultados. Observou-se fraca concordância (3 de 10 comparações pareadas com kappa >0,4), baixa sensibilidade e especificidade e fraco desempenho de ambos os métodos na identificação do E. granulosus em amostras fecais de cães nos laboratórios participantes do estudo. Uma comparação retroativa revelou considerável heterogeneidade dos protocolos de análise laboratorial, o que poderia em parte explicar a fraca concordância entre os resultados. Conclusões. Os resultados deste estudo apontam para a falta de uniformidade no diagnóstico de equinococose canina em toda a Região e indicam possíveis causas para variabilidade. A padronização da análise laboratorial da equinococose canina deve constar do plano de ação para a Iniciativa Regional para Controle da Hidatidose. Outras comparações de amostras fecais de parasitas conhecidos devem ser realizadas.

Self-fluorescent antibiotic MoOx-hydroxyapatite: a nano-theranostic platform for bone infection therapies.

Nowadays, the repair of large-size bone defects represents a huge medical challenge. A line of attack is the construction of advanced biomaterials having multifunctional properties. In this work, we show the creation of biocompatible MoOx-hydroxyapatite nanoparticles (nano-HA/MoOx) that simultaneously exhibit self-activated fluorescence and antibiotic skills. Along this text, we demonstrate that the insertion of molybdenum, an essential trace element, into the non-stoichiometric calcium deficient hydroxyapatite lattice generates intrinsic electronic point defects that exacerbate its epifluorescence blue emission and provokes new red emissions, preserving, always, its bioactivity. Furthermore, these point defects, acting as electron acceptors, stimulate the materials' biological redox status and promote the death of pathogen microorganisms after their direct contact. A putative mechanism, by which bacteria lose electrons from their metabolic circuit that alter the function of their cytoplasmic membrane and potentially die, agrees with our results. Our findings highlight the importance of tuning the electronic communications between biomaterial interfaces and biological units, and support the use of self-fluorescent MoOx-hydroxyapatite nanoparticles as fundamental building blocks for new real-time imaging platforms against bone infection.

Infection of Rat Osteoblasts with Junin Virus Promotes the Expression of Bone Morphogenetic Protein 6, an Osteogenic Differentiation Inducer.

BACKGROUND: The arenavirus Junin virus (JUNV), causative agent of the argentine hemorrhagic fever, is able to modulate several signaling pathways involved in cell survival and multiplication. OBJECTIVES: We aimed to characterize the infection of rat osteoblasts (OBCs) with JUNV and its consequence on the modulation of osteogenic genes expression, thus studying the ability of this virus to induce cell differentiation. In addition, we evaluated the effect of purinergic agonists on viral replication. METHOD: Quantification of infectivity by plaque forming unit (PFU) assay, synthesis of viral proteins by western blot and immunofluorescence, and expression of osteogenic differentiation markers (ODM) by quantitative real-time polymerase chain reaction were employed. RESULTS: Infection of OBCs with JUNV (MOI 0.01 PFU/cell) showed a peak of infectivity, reaching 1.5 × 105 PFU/mL at the second day post-infection (p.i.). A marked restriction in multiplication was detected at day 7 p.i. that did not impair the establishment of a persistent stage of infection in OBCs. Analysis of mRNAs corresponding to ODM such as alkaline phosphatase, bone sialo-protein, and bone morphogenetic proteins (BMPs) 4 and 6 revealed that only the levels of BMP-6 were significantly higher in infected cells. Treatment with the purinergic agonists ATPγS, UTP, ADP, or UDP diminished viral titer and reduced the expression of the viral nucleoprotein. Also, treatment with 10 µM ATPγS reduced the stimulation of BMP-6 expression induced by the infection. CONCLUSIONS: These data demonstrate that JUNV is capable of infecting OBCs and point out BMP-6 as a key factor during this process.

Pilot field trial of the EG95 vaccine against ovine cystic echinococcosis in Rio Negro, Argentina: 8 years of work.

Cystic echinococcosis (CE) is endemic in the Rio Negro province of Argentina. After 30 years of control using praziquantel in dogs the transmission rate to humans and sheep has decreased significantly, however transmission persists. The objective of the study was to assess the inclusion of the EG95 for sheep in the control program and to determine the vaccine's operative feasibility in field conditions. An intervention study was defined in Rio Negro Province in Argentina comprising, in total, an area of 5820 Km2. Lambs received two vaccinations with the EG95 vaccine followed by a single booster injection when the animals were 1-1.5 years of age. Vaccination of lambs born into one trial site was introduced and continued for 8 years. Evidence for Echinococcus granulosus transmission was monitored before and after vaccination by coproantigen ELISA in faecal samples of dog, purgation of dogs to detect E. granulosus worms, necropsy on adult sheep and by ultrasound screening in children of 6-14 years old. 29,323 doses of vaccine were applied between 2009 and 2017, which a vaccination coverage of 80.1%/85.7% (57.3% average for fully vaccinated). Before the introduction of the vaccine 56.3% of the 6-year-old sheep were infected with E. granulosus at necropsy and 84.2% of the farms had infected sheep; 4.3% of the dogs were positive for E. granulosus infection using the arecoline test, and with coproELISA 9.6% of dog fecal samples were positive and 20.3% of the farms had infected dog.After the vaccine was introduced, 21.6% of sheep older than 6 years were found to be infected at necropsy and 20.2% of the farms were found to be infected; in dogs, 4.5% were found positive for E. granulosus using arecoline purgation and with coproELISA 3.7% of samples were positive, with 8.9% of farms having a positive dog. In 2016 only one case of E. granulosus infection was diagnosed by US screening in a 6-14 years old child. Included in the analysis are discussions of difficulties experienced in the field which affected correct vaccine administration as well as social features and practices that may impact on echinococcosis control and the EG95 vaccination program in Rio Negro. Vaccination of sheep with the EG95 vaccine provides a valuable new tool which improves the effectiveness of CE control activities. Vaccination was effective even in a difficult, remote environment where only approximately half the lambs born into the communities were fully vaccinated.

First inter-laboratory comparison of Echinococcus granulosus sensu lato diagnosis in Latin America.

OBJECTIVE: To compare the performance of polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) tests for diagnosing Echinococcus granulosus in dog feces among national reference laboratories in Argentina, Chile, Peru, and Uruguay. METHODS: National laboratories affiliated with the Ministry of Health/Agriculture of each country exchanged panels of 10 positive/negative samples obtained from their regular national surveillance programs in November 2015 - November 2016. All laboratories applied PCR; two also applied ELISA techniques. Sensitivity and specificity were determined for each laboratory and concordance of results among the laboratories was evaluated by Cohen Kappa coefficient. RESULTS: Poor concordance (3 of 10 paired comparisons had values of Kappa > 0.4), low sensitivity and specificity across all laboratories, and poor performance of both techniques in detecting E. granulosus in canine feces was demonstrated in this study. An ex-post comparison of the laboratories' test protocols showed substantial heterogeneity that could partially explain poor concordance of results. CONCLUSION: The results underscore the heterogeneity of canine echinococcosis diagnosis across the region and indicate possible sources of variability. Efforts to standardize canine echinococcosis testing must be included in the plan of action for the Regional Initiative for the Control of Cystic Echinococcosis. Future comparisons with fecal samples of known parasite load are needed.

Multi-drug delivery system based on lipid membrane mimetic coated nano-hydroxyapatite formulations.

Local delivery systems from an osteoconductive biomaterial are suggested as a promising strategy to avoid simultaneously peri-implant traumas and to induce tissue regeneration. In this work, it is detailed the design and construction of a multi-drug delivery formulation based on lipid membrane mimetic coated nano-hydroxyapatite, LMm/nano-HA, as a bone-specific drug delivery approach. The optimal LMm/nano-HA formulation was selected after analysing the lipid/nano-HA interaction by dynamic light scattering (DLS), ζ-potential, transmission electron microscopy (TEM), polarized optical microscopy (POM), differential scanning calorimetry (DSC) and UV-vis spectroscopy. After the initial screening, Ciprofloxacin and Ibuprofen simultaneous -load and -release efficiency from selected LMm/nano-HA was assessed. pH-responsive kinetic profiles of local drug distribution were characterized and compared with currently applied systemic doses. Finally, the systems' biocompatibility and drug released activity were positively validated. The obtained results demonstrated that LMm/nano-HA formulations can represent a valuable multi-modal platform in bone tissue therapies.

Involvement of GSK3/ß-catenin in the action of extracellular ATP on differentiation of primary cultures from rat calvaria into osteoblasts.

Modulation of purinergic receptors play an important role in the regulation of osteoblasts differentiation and bone formation. In this study, we investigated the involvement of the GSK3/ßcatenin signaling in the action of ATPγ-S on osteogenic differentiation of primary cell cultures from rat calvaria. Our results indicate that the cell treatment with 10 or 100 µM ATPγ-S for 96 h increase the cytoplasmic levels of ß-catenin and its translocation to nucleus respect to control. A similar effect was observed after cell treatment with the GSK3 inhibitor LiCl (10 mM). Cell treatments with 4-10 mM LiCl significantly stimulated ALP activity respect to control at 4 and 7 days, suggesting that inhibition of GSK-3 mediates osteoblastic differentiation of rat calvarial cells. Effects comparison between ATP and LiCl shown that ALP activity was significantly increased by 10 µM ATPγ-S and decreased by 10 mM LiCl at 10 day of treatment, respect to control, suggesting that the effect of ATPγ-S was less potent but more persistent than of LiCl in stimulating this osteogenic marker in calvarial cells. Cell culture mineralization was significantly increased by treatment with 10 µM ATPγ-S and decreased by 10 mM LiCl, respect to control. In together, these results suggest that GSK3 inhibition is involved in ATPγ-S action on rat calvarial cell differentiation into osteoblasts at early steadies. In addition such inhibition by LiCl appear promote osteoblasts differentiation at beginning but has a deleterious effect on its function at later steadies as the extracellular matrix mineralization.

Evaluación de echinococcosis canina en la zona de alta montaña en la provincia de Tucumán, Argentina / Evaluation of canine echinococcosis in the high mountain area of province of Tucumán, Argentina / Avaliação de equinococose canina na zona de alta montanha na província de Tucumán, Argentina

La echinococcosis es un serio problema para la salud pública en Argentina. Es una zoonosis producida por el cestode de la familia Taeniidae, Echinococcus granulosus, siendo el perro el principal hospedador definitivo. El objetivo de este trabajo fue evaluar la echinococcosis en perros en la zona de alta montaña de la provincia de Tucumán. La principal actividad económica en esta zona es la cría de ganado para consumo familiar, sobre todo ovino. Las condiciones climáticas, las distancias y la falta de movilidad adecuada hacen difícil el acceso a los servicios de salud y a los establecimientos educativos. Se evaluaron seis parajes en un primer estudio con 103 muestras de materia fecal canina y se encontró que el 39,8% (41/103) fueron positivas. El estudio se repitió en dos localidades después de desparasitar a los perros mensualmente durante dos años. En estas áreas se observó una disminución de muestras con resultado positivo; en Chaquivil la variación fue del 57,1% al 10,0% y en San José de Chasquivil, fue del 47,1% al 11,1%. Estos resultados llevan a la conclusión de que es necesario realizar y planificar las actividades de control. Sería de suma importancia realizar la evaluación en otras localidades.

Canine echinococcosis is a serious public health problem in Argentina. It is a parasite zoonosis produced by a cestode of the Taeniidae family, Echinococcus granulosus, the dog being the principal definite host. The objective of this work was to evaluate echinococcosis in dogs in the high mountain area of the province of Tucumán. The main economic activity in this area is livestock breeding, mostly sheep, for family consumption. Climate conditions, distances and the lack of adequate mobility make it difficult to access to health services and educational facilities. Six places of the region were evaluated. The first study was carried out with 103 samples of canine faeces, 39.8% (41/103) being positive. The study was repeated in two localities, after deworming dogs monthly for two years. In these areas, a decrease of positive specimens was obtained: in Chaquivil, variation was 57.1% to 10.0% and in San José de Chasquivil, it was 47.1% to 11.1%. These results lead to the conclusion that it would be necessary to conduct and plan control activities. The assessment of other locations would be of utmost importance.

A equinococose é um problema sério para a saúde pública na Argentina. É uma zoonose produzida pelo cestode da família Taeniidae, Echinococcus granulosus, sendo o cão o principal hospedeiro definitivo. O objetivo deste trabalho foi avaliar a equinococose em cães na zona de alta montanha da Província de Tucumán. A atividade econômica principal nesta zona é a criação de gado para consumo familiar, principalmente o ovino. As condições climáticas, as distâncias e a falta da mobilidade adequada tornam difícil o acesso aos serviços da saúde e aos estabelecimentos educacionais. Seis lugares da região foram avaliados num primeiro estudo com 103 amostras da matéria fecal canina, sendo que 39.8% (41/103) foram positivas. O estudo foi repetido em duas localidades, depois de desverminar os cães mensalmente durante dois anos. Nessas áreas foi observada uma diminuição de amostras com resultado positivo; em Chaquivil a variação foi de 57.1% para 10.0% e em San José de Chasquivil, foi 47.1% para 11.1%. Estes resultados levam à conclusão de que é necessário realizar e planejar as atividades do controle. Seria de extrema importância fazer a avaliação em outras localidades.

Effect of Combined Action of Extracellular ATP and Elevated Calcium on Osteogenic Differentiation of Primary Cultures From Rat Calvaria.

The in vitro osteogenic differentiation has been intensively studied. However, it is not yet clear precisely how osteogenesis can be optimized. Changes in extracellular Ca(2+) concentration ([Ca(2+) ]e ), as well as modulation of purinergic receptors play an important role in the regulation of osteoblasts differentiation and bone formation. In this study, we investigated the effects of a combined treatment of ATPγ-S and high [Ca(2+) ]e (5.35 mM) on osteogenic differentiation and function of primary cell cultures from rat calvaria. Our results indicate that ATPγ-S stimulates cell transition from the G0 to S phase of cell cycle, involving the PI3K signaling pathway. Treatment with 10 or 100 µM ATPγ-S and [Ca(2+) ]e (ATP-[Ca(2+) ]e ) for 48 h increases cell number significantly above the control. ATPγ-S treatment in osteogenic medium containing [Ca(2+) ]e stimulates the gene expression of BMP-4, BMP-5, and OPN at 16, 48, and 72 h, respectively, above control. In same conditions, treatment for 6 days with 10 µM UTP or 100 µM UDP significantly increased the ALP activity respect to control. Cells grown in osteogenic medium showed a statistically significant increase in calcium deposits at 15 and 18 days, for 10 µM ATPγ-S treatment, and at 18 and 22 days, for [Ca(2+) ]e treatment, respect to control but ATP-[Ca(2+) ]e treatment shown a significant greater mineralization at 15 days respect to ATPγ-S, and at 18 days respect to both agonists. In conclusion, we demonstrated that an osteogenic medium containing 10 µM ATPγ-S and 5.35 mM [Ca(2+) ]e enhance osteogenesis and mineralization by rat primary calvarial cells cultures. J. Cell. Biochem. 117: 2658-2668, 2016. © 2016 Wiley Periodicals, Inc.

Estudio de infestación de caninos con Echinococcus granulosus en la provincia de La Rioja, Argentina / Study of infestatioon of dogs with Echinococcus granulosus in the province of La Rioja, Argentina

Este trabajo fue realizado en la provincia de La Rioja, ubicada en el noroeste argentino. El objetivo fue estimar el porcentaje de heces de caninos con presencia de antígenos de Echinococcus sp. en las distintas regiones de la provincia. Se tomaron un total de 269 muestras de materia fecal seca de caninos, que fueron analizadas por la técnica de copro-ELISA. La zona más afectada fue la zona IV, que contó con un 30,5% de muestras positivas. La zona I, que corresponde al Departamento Capital, tuvo un 12% de positividad. En el resto de las zonas, los porcentajes variaron entre el 11,4 y el 14,8%. Este es el primer estudio en la provincia sobre la existencia de la enfermedad en caninos. La falta de estrategias para el control de la equinococosis ha permitido la dispersión de la enfermedad

This work was conducted in the province of La Rioja, located in northwestern Argentina. The aim of this study was to estimate the percentage of dog feces showing the presence of antigens of Echinococcus sp. in different regions of the province. A total of 269 samples of dried canine stool were taken, which were analyzed by the copro-ELISA technique. The most affected area was zone IV, which had 30.5% of positive samples. Zone I corresponding to the Capital Department of the province had 12% of positivity. In other areas, the percentages ranged between 11.4% and 14.8%. This is the first study in the province of La Rioja on the existence of this disease in dogs. The lack of control strategies has allowed the spread of echinococcosis

[Study of infestation of dogs with Echinococcus granulosus in the province of La Rioja, Argentina]. / Estudio de infestación de caninos con Echinococcus granulosus en la provincia de La Rioja, Argentina.

This work was conducted in the province of La Rioja, located in northwestern Argentina. The aim of this study was to estimate the percentage of dog feces showing the presence of antigens of Echinococcus sp. in different regions of the province. A total of 269 samples of dried canine stool were taken, which were analyzed by the copro-ELISA technique. The most affected area was zone IV, which had 30.5% of positive samples. Zone I corresponding to the Capital Department of the province had 12% of positivity. In other areas, the percentages ranged between 11.4% and 14.8%. This is the first study in the province of La Rioja on the existence of this disease in dogs. The lack of control strategies has allowed the spread of echinococcosis.

Bone-repair properties of biodegradable hydroxyapatite nano-rod superstructures.

Nano-hydroxyapatite (nano-HAp) materials show an analogous chemical composition to the biogenic mineral components of calcified tissues and depending on their topography they may mimic the specific arrangement of the crystals in bone. In this work, we have evaluated the potential of four synthesized nano-HAp superstructures for the in vitro conditions of bone-repair. Experiments are underway to investigate the effects of the material microstructure, surface roughness and hydrophilicity on their osseo-integration, osteo-conduction and osteo-induction abilities. Materials were tested in the presence of both, rat primary osteoblasts and rabbit mesenchymal stem cells. The following aspects are discussed: (i) cytotoxicity and material degradation; (ii) rat osteoblast spreading, proliferation and differentiation; and (iii) rabbit mesenchymal stem cell adhesion on nano-HAp and nano-HAp/collagen type I coatings. We effectively prepared a material based on biomimetic HAp nano-rods displaying the appropriate surface topography, hydrophilicity and degradation properties to induce the in vitro desired cellular responses for bone bonding and healing. Cells seeded on the selected material readily attached, proliferated and differentiated, as confirmed by cell viability, mitochondrial metabolic activity, alkaline phosphatase (ALP) activity and cytoskeletal integrity analysis by immunofluorescence localization of alpha-smooth muscle actin (α-SMA) protein. These results highlight the influence of material's surface characteristics to determine their tissue regeneration potential and their future use in engineering osteogenic scaffolds for orthopedic implants.