Validity and realiability of a new optoelectronic system for measuring active range of motion of upper limb joints in asymptomatic and symptomatic subjects

©2019. This manuscript version is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Published, version of a Published Work that appeared in final form in Journal of Clinical Medicine . To access the final edited and published work see https://doi.org/10.3390/jcm8111851The aim of this study was to evaluate the validity of the Veloflex infrared dynamic angle-meter (Veloflex-IDA) and the intra- and inter-rater reliability when measuring the ranges of motion (ROMs) of the upper limb joints. Thirty-five healthy and 20 symptomatic participants were evaluated. Twelve upper limb movements were measured in two sessions with the Veloflex-IDA, which is a device composed of a camera that tracks the trajectory of retro-reflective markers. In addition, a goniometer was used in the first session to evaluate concurrent validity. Validity and agreement were evaluated by Pearson correlation coefficient (r) and Bland-Altmann plots. Intra- and inter-rater reliability were evaluated using intra-class correlation (ICC), standard error of measurement (SEM), and minimal detectable change (MDC). Both instruments showed excellent correlation for all movements (r range from 0.992 to 0.999). The intra- and inter-rater reliability were excellent (ICC range from 0.95 to 0.99 and 0.90 to 0.98, respectively). Intra-rater reliability showed SEMs <1.38% and <5.19% and inter-rater reliability SEMs <2.26% and <5.22% for asymptomatic and symptomatic, respectively. Veloflex-IDA is a valid and reliable alternative to measure the upper limb joints' ROM and it can be used in clinical practice and research after basic training

Conditional genome engineering reveals canonical and divergent roles for the Hus1 component of the 9-1-1 complex in the maintenance of the plastic genome of Leishmania.

Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9- RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability, and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9-1-1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryote

Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death.

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death

Cooperation between Apoptotic and Viable Metacyclics Enhances the Pathogenesis of Leishmaniasis

Mimicking mammalian apoptotic cells by exposing phosphatidylserine (PS) is a strategy used by virus and parasitic protozoa to escape host protective inflammatory responses. With Leishmania amazonensis (La), apoptotic mimicry is a prerogative of the intramacrophagic amastigote form of the parasite and is modulated by the host. Now we show that differently from what happens with amastigotes, promastigotes exposing PS are non-viable, non-infective cells, undergoing apoptotic death. As part of the normal metacyclogenic process occurring in axenic cultures and in the gut of sand fly vectors, a sub-population of metacyclic promastigotes exposes PS. Apoptotic death of the purified PS-positive (PSPOS) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PSPOS metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNELPOS promastigotes were detected only in the anterior midgut to foregut boundary of infected sand flies. Interestingly, caspase inhibitors modulated parasite death and PS exposure, when added to parasite cultures in a specific time window. Efficient in vitro macrophage infections and in vivo lesions only occur when PSPOS and PS-negative (PSNEG) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PSNEG promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PSPOS apoptotic sub-population inhibited host macrophage inflammatory response. PS exposure and macrophage inhibition by a subpopulation of promastigotes is a different mechanism than the one previously described with amastigotes, where the entire population exposes PS. Both mechanisms co-exist and play a role in the transmission and development of the disease in case of infection by La. Since both processes confer selective advantages to the infective microorganism they justify the occurrence of apoptotic features in a unicellular pathogen

Simultaneous transcriptional profiling of Leishmania major and its murine macrophage host cell reveals insights into host-pathogen interactions

Parasites of the genus Leishmania are the causative agents of leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The life cycle of Leishmania parasites is split between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must evade or deactivate the host's innate and adaptive immune responses in order to survive and replicate. We performed transcriptome profiling using RNA-seq to simultaneously identify global changes in murine macrophage and L. major gene expression as the parasite entered and persisted within murine macrophages during the first 72 h of an infection. Differential gene expression, pathway, and gene ontology analyses enabled us to identify modulations in host and parasite responses during an infection. The most substantial and dynamic gene expression responses by both macrophage and parasite were observed during early infection. Murine genes related to both pro- and anti-inflammatory immune responses and glycolysis were substantially upregulated and genes related to lipid metabolism, biogenesis, and Fc gamma receptor-mediated phagocytosis were downregulated. Upregulated parasite genes included those aimed at mitigating the effects of an oxidative response by the host immune system while downregulated genes were related to translation, cell signaling, fatty acid biosynthesis, and flagellum structure. The gene expression patterns identified in this work yield signatures that characterize multiple developmental stages of L. major parasites and the coordinated response of Leishmania-infected macrophages in the real-time setting of a dual biological system. This comprehensive dataset offers a clearer and more sensitive picture of the interplay between host and parasite during intracellular infection, providing additional insights into how pathogens are able to evade host defenses and modulate the biological functions of the cell in order to survive in the mammalian environment.https://doi.org/10.1186/s12864-015-2237-

Capacidad discriminante de los hallazgos en tomografía para la identificación de lesiones pulmonares por infección con nocardia

Objective: Evaluate the diagnostic value of some findings in thoracic computed tomography, in patients with confirmed pulmonary nocardiosis, so that, taken together, they may help distinguish it from other conditions that may show superimposable radiological findings. Material and methods: Subjects with microbiological diagnosis of nocardiosis in our hospital over a 5-year period were selected. The findings observed in tomography were collected and compared with those observed in three comparison groups made up by pathologies which produce superimposable image manifestations (tuberculosis, pulmonary neoplasms, and pneumonias). We sought to establish a model that would help distinguish, based on image findings, pulmonary nocardiosis from other groups. Results: We established a model which combines three variables (age, adenopathies, and heterogeneous size of nodules) and obtained a dis- criminatory capacity of 79 % compared with other alternative diagnoses. Conclusions: although the findings in image techniques are not specific for pulmonary nocardiosis, the grouping of findings in our model, in a proper clinical context, may be useful in establishing a provisional diagnosis of pulmonary nocardiosis, to the detriment of other possibili- ties, and help manage therapeutic approaches pending microbiological confirmation.Objetivo: evaluar el valor diagnóstico de algunos hallazgos en tomo - grafía computada de tórax, en pacientes con nocardiosis pulmonar confirmada, con el fin de que en conjunto permitan diferenciarla de otras afecciones que pueden mostrar hallazgos radiológicos superponibles. Material y métodos: se seleccionaron los sujetos con diagnóstico micro - biológico de nocardiosis en nuestro hospital en un periodo de 5 años. Se recogieron los hallazgos observados en tomografía y se compararon con los observados en tres grupos de comparación constituidos por patologías que producen manifestaciones de imagen superponibles (tuberculosis, neoplasias pulmonares y neumonías). Se intentó estable- cer un modelo que permitiera diferenciar, por imagen, la nocardiosis pulmonar de los otros grupos. Resultados: establecimos un modelo que combina tres variables (edad, adenopatías y tamaño heterogéneo de los nódulos) y obtuvimos una ca - pacidad discriminativa de 79% frente a otros diagnósticos alternativos. Conclusiones: a pesar de que los hallazgos en técnicas de imagen no son específicos para nocardiosis pulmonar, la agrupación de los ha- llazgos de nuestro modelo, en un contexto clínico adecuado, puede ser de utilidad a la hora de establecer un diagnóstico provisional de nocardiosis pulmonar, en detrimento de otras posibilidades, y permite gestionar conductas terapéuticas mientras se espera la confirmación microbiológica

Modificación en el patrón de uso de anfotericina B no convencional tras la puesta en marcha de una intervención formativa en el Hospital Clínico San Carlos de Madrid

Background: Amphotericin B is the treatment of choice for systemic fungal infections. Among the different AB formulations available, the lipid forms appear to have a better profile of reliability, however, their cost is noticeably higher. In 1999 (pre-initiative period) an evaluation of the quality of the prescription of these preparations was made in our hospital, which revealed that they were not being used to best advantage and were responsible for generating a significant unnecessary expenditure. As a result of this, an information initiative was implemented with respect to the prescribing physicians for the purpose of reducing the inappropriate use of AB. Method: The quality of 100 prescriptions was evaluated prospectively, according to the standards of use of Amphotericin B established in the hospital. Following each evaluation, a pharmacologist personally handed over to each prescribing physician a set of rules governing the use of the Amphotericin B, discussing the indication and recommending the best alternative in each case. In order to measure the impact of this initiative, the appropriateness of the prescriptions during this period was compared with the pre-initiative period. Results: The percentage of inappropriate prescriptions dropped from 58% to 21% following the implementation of the initiative. Likewise, a 33-million-peseta reduction in the total expenditure was achieved in 15 months as well as a savings of 24 million in inappropriate prescriptions. Conclusions: The information initiative improved the quality of the prescribing of preparations of Amphotericin B associated with lipids and considerably reduced the unnecessary expense associated with Amphotericin B misuse in our hospital.Fundamento: La anfotericina B es el tratamiento de elección para las infecciones fúngicas sistémicas. Dentro de ellas, las formas lipídicas parecen tener un mejor perfil de seguridad, sin embargo el coste es llamativamente superior. En 1999 (periodo pre-intervención) se realizó en nuestro hospital una evaluación de la calidad de la prescripción de estos preparados que demostró que su uso no era óptimo y ocasionó un gasto innecesario importante. Como consecuencia de ello se puso en marcha una intervención formativa sobre los prescriptores con el objetivo de reducir el uso inapropiado de AB. Método: Se evaluó prospectivamente la calidad de 100 prescripciones, según las normas de uso de Anfotericina B vigentes en el hospital. Tras cada evaluación un farmacólogo entregó personalmente a cada prescriptor unas normas de uso del antibiótico, discutiendo la indicación y recomendando la mejor alternativa en cada caso. Para medir el impacto de esta intervención se comparó la adecuación de las prescripciones en este periodo frente al periodo pre-intervención. Resultados: El porcentaje de prescripciones inadecuadas se redujo del 58 % al 21 % tras la puesta en marcha de la intervención. Así mismo, se produjo una reducción en el gasto total de 33 millones de pesetas en 15 meses y un ahorro de 24 millones en prescripciones inadecuadas. Conclusiones: La intervención formativa mejoró la calidad de la prescripción de preparados de AB asociada a lípidos y redujo considerablemente el gasto innecesario de AB en nuestro hospital

Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5

Background: Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced. Results: We have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (ΔLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of ΔLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving ΔLicpa::CPA) was sufficient to complement the reduced infectivity of both ΔLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone ΔLicpaC1::CPA compared with the CPA-deficient mutant ΔLicpaC1. Conclusion: The data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the ΔLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters