Early Leishmania infectivity depends on miR-372/373/520d family-mediated reprogramming of polyamines metabolism in THP-1-derived macrophages.

Leishmania amazonensis is a protozoan that primarily causes cutaneous leishmaniasis in humans. The parasite relies on the amino acid arginine to survive within macrophages and establish infection, since it is a precursor for producing polyamines. On the other hand, arginine can be metabolized via nitric oxide synthase 2 (NOS2) to produce the microbicidal molecule nitric oxide (NO), although this mechanism does not apply to human macrophages since they lack NOS2 activity. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at posttranscriptional levels. Our previous work showed that mmu-miR-294 targets Nos2 favoring Leishmania survival in murine macrophages. Here, we demonstrate that human macrophages upregulate the hsa-miR-372, hsa-miR-373, and hsa-miR-520d, which present the same seed sequence as the murine mmu-miR-294. Inhibition of the miR-372 impaired Leishmania survival in THP-1 macrophages and the effect was further enhanced with combinatorial inhibition of the miR-372/373/520d family, pointing to a cooperative mechanism. However, this reduction in survival is not caused by miRNA-targeting of NOS2, since the seed-binding motif found in mice is not conserved in the human 3'UTR. Instead, we showed the miR-372/373/520d family targeting the macrophage's main arginine transporter SLC7A2/CAT2 during infection. Arginine-related metabolism was markedly altered in response to infection and miRNA inhibition, as measured by Mass Spectrometry-based metabolomics. We found that Leishmania infection upregulates polyamines production in macrophages, as opposed to simultaneous inhibition of miR-372/373/520d, which decreased putrescine and spermine levels compared to the negative control. Overall, our study demonstrates miRNA-dependent modulation of polyamines production, establishing permissive conditions for intracellular parasite survival. Although the effector mechanisms causing host cell immunometabolic adaptations involve various parasite and host-derived signals, our findings suggest that the miR-372/373/520d family may represent a potential target for the development of new therapeutic strategies against cutaneous leishmaniasis.

Serological and molecular survey of canine distemper virus in red foxes (Vulpes vulpes): Exploring cut-off values and the use of protein A in ELISA tests.

The wide distribution and ecological plasticity of the red fox (Vulpes vulpes) make it a potential reservoir for many infectious diseases shared with domestic and wild carnivores. One of such diseases is canine distemper, which is caused by an RNA virus and its main domestic reservoir is the dog. However, other carnivores can also participate in its maintenance, as shown by the recent upsurge of reported cases in wildlife in many parts of the world, and by the fact that red foxes may act as true reservoirs for canine distemper virus (CDV). The lack of validated serological tests for wildlife or other non-target species may be a handicap for monitoring this virus. In this study, serological assays were compared in 147 red fox sera using a commercial ELISA validated for its use in dogs and a non-specific modified ELISA with Protein A peroxidase conjugate to detect bound antibodies. In addition, the presence of CDV RNA in brain, spleen, lung, and liver samples from 144 foxes was investigated by a RT-qPCR. Through the comparison of the results of both ELISAs and the use of a finite mixture model of the optical density values obtained by both techniques, we adjusted the cut-off point of the commercial ELISA to obtain the seroprevalence in foxes. The overall seroprevalence detected was 53.7% (79/147) and 57.1% (84/147) by the commercial and modified ELISA, respectively, with a moderate agreement according to Cohen's Kappa statistic (κ = 0.491, z = 5.97, p < 0.0001). CDV RNA was detected in 30 out of 144 foxes, which resulted in 20.8% of CDV-infected foxes. At individual level, the results obtained by relating the serological status and the presence/absence of RNA in different organs were explained in terms of the pathogenesis of the infection. Our results highlight the convenience of adjusting the cut-off point when using an ELISA assay developed in domestic dogs for its use in foxes. Moreover, Protein A is confirmed to be a good alternative to be used in red foxes, presenting a good reactivity towards its IgG.

Flying across Europe: the case of the spread of Chaunocephalus ferox on a black stork (Ciconia nigra).

The annual migration of birds involves a very large number of inter-continental and intra-continental movements in which thousands of bird species participate. These migrations have been associated with the spread of pathogens worldwide, including bacteria, viruses and parasites. This study describes the case of a black stork (Ciconia nigra) that was ringed at the nest in Latvia and died five months later in the south-east of the Iberian Peninsula. Post-mortem examination revealed that the cause of death was electrocution. In addition, a massive infection by the trematode Chaunocephalus ferox (Digenea: Echinostomatidae) causing severe granulomatous lesions throughout the small intestine was detected. This is the first report of C. ferox infection in a black stork in the Iberian Peninsula, a trematode that, due to the severe lesions it causes, can affect the health of C. ferox-infected wild birds, particularly in severely infected long-distance migrants. The dispersal of platyhelminths associated with migratory birds is discussed. After the ringing at the nest, the black stork was sighted in Central Europe one month before its capture, and the trematodes found by necropsy were mostly mature adults. Consequently, we estimate that this juvenile animal acquired the infection during its migration in a European area other than the Iberian Peninsula, evidencing a long-distance parasite spread through its migratory host. Our study highlights that bird ringing can be used to understand the epidemiological implications that bird migratory behaviour may have on the dispersal of parasites.

Assessing DESS solution for the long-term preservation of nematodes from faecal samples.

Preservation of biological samples is a relevant issue for many scientific disciplines. Although traditional preservers, such as formaldehyde or ethanol, imply major disadvantages related to health risks, DNA degradation and distortion of structures, they are widely used. Hence, the search for viable alternatives preserving morphometry and genetics seems necessary. Here we assess the suitability of DESS solution to preserve adult nematodes and their eggs in faeces. Concretely, faecal samples of terrestrial tortoises with oxyurids were used to: (i) compare the 1-month storage efficacy of eggs from different conservation protocols (faeces without preserver at -20 °C, faeces with DESS solution at room temperature, faeces with DESS solution at -20 °C and faeces with ethanol 70% at room temperature); (ii) address morphological nematode identification after 2 years of storage with DESS. We also corroborated that nematode DNA remained viable after 2 years. Overall, our results showed that DESS solution at room temperature is an advisable alternative to conserve both parasite eggs and adult nematodes for morphological identification and genetic purposes. It also offers the advantages of being low-cost, safe and suitable for fieldwork conditions and shipments without refrigeration for nematode preservation.

First report of a hawfinch (Coccothraustes coccothraustes) parasitized by Harpirhynchus nidulans in the Iberian Peninsula.

Some avian Harpirhynchidae mites live under the skin and develop cutaneous cysts. Despite the obvious lesions that these parasites can produce, little is currently known about the behavioural disturbances that cyst-forming mites may cause in infected wild birds. We report an infection by Harpirhynchidae mites in a hawfinch (Coccothraustes coccothraustes) in southeast Spain. The bird was easily captured due to its inability to fly. During clinical examination it was found to have multiple severe traumatic injuries, possibly due to a blow or a fall, as result of which the bird was euthanized. At necropsy, the hawfinch was found to be in good body condition. Two yellowish and friable mite-filled cysts were detected in the subalar region of both wings. Mites were morphologically identified as Harpirhynchus nidulans, and histological analysis of the cystic lesions was also performed. This is the first time that the occurrence of a hawfinch infected by H. nidulans in the Iberian Peninsula has been reported.

Density assessment and reporting for Phlebotomus perniciosus and other sand fly species in periurban residential estates in Spain.

Green periurban residential areas in Mediterranean countries have flourished in the last decades and become foci for leishmaniasis. To remedy the absence of information on vector ecology in these environments, we examined phlebotomine sand fly distribution in 29 sites in Murcia City over a 3-year period, including the plots of 20 detached houses and nine non-urbanized sites nearby. We collected 5,066 specimens from five species using "sticky" interception and light attraction traps. The relative frequency of the main Leishmania infantum vector Phlebotomus perniciosus in these traps was 32% and 63%, respectively. Sand fly density was widely variable spatially and temporally and greatest in non-urbanized sites, particularly in caves and abandoned buildings close to domestic animal holdings. Phlebotomus perniciosus density in house plots was positively correlated with those in non-urbanized sites, greatest in larger properties with extensive vegetation and non-permanently lived, but not associated to dog presence or a history of canine leishmaniasis. Within house plots, sand fly density was highest in traps closest to walls. Furthermore, the study provides a guideline for insect density assessment and reporting and is envisioned as a building block towards the development of a pan-European database for robust investigation of environmental determinants of sand fly distribution.

Nasopharyngeal bot flies in red deer (Cervus elaphus) from southern Spain.

The nasopharyngeal bot flies Pharyngomyia picta and Cephenemyia auribarbis (Diptera: Oestridae) are parasites that have an impact on the health of wild ruminants. Little is known about their biological and epidemiological characteristics in multi-host habitats such as the Sierra de Cazorla, Segura y Las Villas Natural Park (CNP), in south-east Spain. This paper describes the main epidemiological features of bot fly larvae parasitising the population of red deer in CNP during 2003-2005. The results reveal an overall bot fly prevalence of 37.5%, with a P. picta and C. auribarbis co-infection rate of 12.5%. Although the statistical analyses were not significant(p > 0.05), a higher prevalence in males and in calves was observed, which has to be explained from a multifactorial viewpoint. Unfavourable climatic conditions during January induced the overwintering of larvae inside the host and, accordingly, a higher prevalence of the first instar (L1) was detected, while a higher prevalence of L2 and L3 was recorded in February and March. Further studies are needed to investigate in more detail the environmental characteristics that influence the chronobiology of bot flies in south-east Spain.

Les mouches nasopharyngées Pharyngomyia picta et Cephenemyia auribarbis (Diptera : Oestridae) sont des parasites qui affectent la santé des ruminants sauvages. Leurs caractéristiques biologiques et épidémiologiques au sein d'habitats tels que le Parc naturel des Sierras de Cazorla, Segura y las Villas (CNP) dans le sud-est de l'Espagne, où cohabitent de nombreuses espèces hôtes, sont mal connues. Les auteurs présentent les résultats d'une étude conduite de 2003 à 2005 visant à décrire les principales caractéristiques épidémiologiques de l'infestation par des larves de mouches dans les populations de cerfs élaphes du CNP. D'après les résultats de l'étude, la prévalence globale des mouches s'élève à 37,5 % avec un taux de co-infestation par P. picta et C. auribarbis atteignant 12,5 %. Bien que les analyses statistiques n'aient pas donné de résultats significatifs (p < 0,05), une prévalence plus élevée a été observée chez les mâles et les chevreuils, phénomène qui doit être expliqué dans une perspective multifactorielle. Les conditions climatiques défavorables durant le mois de janvier ont prolongé l'hivernage des larves au sein de l'hôte, ce qui s'est traduit par une prévalence plus élevée du premier stade larvaire (L1), tandis qu'en février et en mars les stades L2 et L3 étaient les plus fréquents. Des études complémentaires devront être menées afin d'élucider plus en détail les facteurs environnementaux susceptibles d'influer sur la chronobiologie de ces mouches dans le sud-est de l'Espagne.

Las moscas causantes de miasis nasofaríngea Pharyngomyia picta y Cephenemyia auribarbis (orden Diptera, familia Oestridae) son parásitos perjudiciales para la salud de los rumiantes salvajes. Poco se sabe acerca de sus características biológicas y epidemiológicas en hábitats donde hay múltiples hospedadores, como es el caso del Parque Natural de las Sierras de Cazorla, Segura y Las Villas (CNP), situado en el sudeste de España. Los autores describen las principales características epidemiológicas que afectan a las larvas de moscas de la miasis que parasitan a la población de ciervos del CNP según un estudio realizado desde 2003 hasta 2005. Los resultados revelan una prevalencia global de estas moscas del 37,5%, con una tasa de coinfección por P. picta y C. auribarbis del 12,5%. Aunque los análisis estadísticos no arrojaron un resultado significativo (p > 0,05), se observó una prevalencia superior en machos y ciervos jóvenes, hecho que debe explicarse desde una perspectiva multifactorial. Las desfavorables condiciones climáticas predominantes en enero propiciaron la diapausa invernal de las larvas en los hospedadores, lo que a su vez explica que se detectase una mayor prevalencia de larvas de primer estadio (L1), mientras que en febrero y marzo se registró una mayor prevalencia de larvas de segundo y tercer estadio (L2 y L3). Se requieren más investigaciones para estudiar con mayor detalle las características ambientales que influyen en la cronobiología de las moscas causantes de miasis en el sudeste de España.

Real-world data on the efficacy and safety of weekly oral vinorelbine in breast cancer patients previously treated with anthracycline or taxane-based regimens.

PURPOSE: To evaluate the efficacy and safety of oral weekly vinorelbine 60 mg/m2 for metastatic breast cancer (MBC) in patients previously treated with anthracyclines or taxanes in routine clinical practice. MATERIALS AND METHODS: Fifty-five patients were enrolled in a prospective multicentre study conducted in Spain. Women ≥ 18 years of age with locally advanced breast cancer who were not candidates for surgical treatment with a radical intention or patients with stage IV disease, and who had received a prior taxane or anthracycline regimen were eligible for participation. RESULTS: Median age was 67 years. Median progression-free survival was 3.7 months (95% CI 2.5-4.9), median overall survival 10 months (95% CI 6.6-13.5), and overall response rate and clinical benefit rate were 29.1% and 49.1%, respectively. Main grade 3 and 4 toxicities were neutropenia 9.1%, febrile neutropenia 3.6% and constipation 3.6%. In total, 86% of the patients received complete treatment without delays or dose reduction. Moreover, HER2-positive patients who received oral vinorelbine concomitantly with trastuzumab showed better response (complete response: HER2-positive 14.3% vs. HER2-negative 0%; partial response: HER2-positive 42.9% vs. HER2-negative 25.6%; p = 0.008), better disease control rate (HER2-positive 100% vs. HER2-negative 46.2%; p = 0.011), and better values for the remaining analysed variables than HER2-negative patients. CONCLUSION: Our study provides real-world data on the use of oral weekly vinorelbine, which proves an effective and well-tolerated regimen for MBC patients previously treated with taxanes or anthracyclines. Patients with HER2-positive disease could also benefit from this treatment in combination with trastuzumab.

Plastinated macroparasites, an alternative resource for use in practical lessons.

The plastination technique is a recently developed option to preserve biological tissues widely used in human and veterinary anatomy. This process allows one to obtain real, safe, clean, dry, resistant and permanently stable material without any additional treatment or maintenance. All these advantages are associated with the rapid expansion of plastination during recent decades in a high number of educational institutions. However, the studies of parasite conservation through plastination are scarce, although the material obtained has a high quality, as prior references have shown. In this study, 141 students of veterinary medicine and pharmacy university degrees used, in a blind study, plastinated specimens (experimental group) or wet specimens (control group) during three consecutive practical sessions. Afterwards all the students completed anonymous questionnaires on both knowledge and satisfaction. The results demonstrated that plastinated parasites are a valid and satisfactory alternative to be used as a teaching/learning tool during practical lessons. Moreover, with regard to the knowledge of morphological structures, the results from the questionnaire of assimilated knowledge showed no differences between the use of plastinated and formaldehyde-preserved parasites.

La technique de plastination est une méthode assez récente de préservation des tissus biologiques qui est désormais largement utilisée en anatomie humaine et vétérinaire. Ce procédé permet d'obtenir des préparations anatomiques réalistes, sûres, sèches, résistantes et stables qui ne nécessiteront pas de traitement ni d'entretien ultérieurs pour préserver leur durabilité. Ces avantages expliquent que le recours à la plastination se soit rapidement développé au cours des dernières décennies dans nombre d'institutions d'enseignement. Néanmoins, rares sont les études portant sur la préservation des parasites par plastination, bien que les spécimens obtenus lors de travaux antérieurs aient été jugés de très bonne qualité. Les auteurs présentent les résultats d'une enquête menée auprès de 141 étudiants préparant leur diplôme de médecine vétérinaire ou de pharmacie, à qui ont été distribués en aveugle des spécimens plastinés (groupe expérimental) et des spécimens conservés dans une solution liquide (groupe de contrôle) lors de trois séances consécutives de travaux pratiques. Il a ensuite été demandé aux étudiants de remplir anonymement un questionnaire portant sur les connaissances acquises lors de ces séances et sur leur degré de satisfaction. Les résultats ont montré que les parasites plastinés constituent une alternative valable et satisfaisante en tant que matériel pédagogique et d'entraînement pendant les leçons pratiques. Par ailleurs, s'agissant de l'étude des structures morphologiques, les résultats du questionnaire sur les connaissances acquises lors des travaux pratiques n'ont pas fait apparaître de différences entre les étudiants ayant travaillé sur des parasites plastinés et ceux ayant travaillé avec des parasites conservés dans le formol.

La técnica de la plastinación es un método de reciente aparición para conservar tejidos biológicos, utilizado con gran frecuencia en anatomía humana y veterinaria. Se trata de un proceso que permite obtener material real, inocuo, limpio, seco, resistente y con una estabilidad permanente sin necesidad de ninguna otra operación de tratamiento o mantenimiento. Todas estas ventajas explican que en los últimos decenios la técnica se haya extendido con rapidez por muy numerosas instituciones de enseñanza. Aun así, hay escasos estudios sobre la conservación de parásitos por plastinación, pese a que el material obtenido, como se desprende de referencias anteriores, es de gran calidad. Los autores describen un estudio ciego en el que 141 estudiantes universitarios en farmacia y medicina veterinaria emplearon muestras plastinadas (grupo experimental) o muestras húmedas (grupo de control) en tres clases prácticas consecutivas, tras lo cual todos los estudiantes respondieron anónimamente a un cuestionario que evaluaba tanto sus conocimientos como su nivel de satisfacción. Los resultados demostraron que los parásitos plastinados constituyen una alternativa válida y satisfactoria como herramienta para impartir y/o recibir clases prácticas. Además, desde el punto de vista del conocimiento de estructuras morfológicas, los resultados del cuestionario sobre conocimientos asimilados no pusieron de manifiesto diferencia alguna entre el uso de parásitos plastinados y el de parásitos conservados en formaldehído .

Clinical and para-clinical description of a novel mutation for Schnyder dystrophy in a French family.

INTRODUCTION: The objective of this article is to describe the evolution of Schnyder dystrophy in 3 related patients of different ages and to highlight the discovery of a new mutation unidentified until now. CASE REPORT: We present a series of 3 cases, all first-degree relatives with no suggestion of consanguinity, of different ages (30, 40 and 59 years) and two distinct generations (mother and children). Slit lamp examination revealed the same lesions in our three patients: an early-onset corneal arcus senilis, central corneal deposits, and a gray stromal haze in the two oldest subjects. The older the patient, the more numerous and dense were these lesions. The various anterior segment OCTs showed an increase in the number of hyperreflective opacities in the anterior stroma and, in the older subject, the appearance of many posterior shadows. Monitoring of pachymetry by Pentacam® showed progressive age-related thickening. All three patients had dyslipidemia treated with statins or diet alone. In our case we proposed treatment only to subject A because of the significant impact on her visual acuity. DISCUSSION: Numerous clinical, para-clinical and genetic descriptions of this disease are found in the literature. Schnyder dystrophy is rare but not unheard of and may be discovered fortuitously or in the setting of decreased visual acuity. Genetic analysis of our family revealed a mutation of the UBIAD1 gene not described in the literature. UBIAD1 encodes the protein domain-containing UbiA prenyltransferase 1 which converts vitamin K1 into K2 and is involved in the cholesterol synthesis pathway. In the case of a mutation, it is no longer functional, leading to the accumulation of cholesterol crystals. Given the clinical context and the presence of this variant of the reference sequence in all relatives, its pathogenesis is strongly suspected in our family. The originality of our article is to present the progression of the same pathology in 3 patients with the same mutation at different ages and degrees of severity. This notion of progressive worsening and the need to treat late in the majority of cases are found in literature. CONCLUSION: The discovery of a new variant within the UBAID1 gene suggests its pathogenesis in view of the clinical features available to us. The dystrophy is initially asymptomatic before the high number of deposits becomes disabling.

Epidemiological and genetic studies suggest a common Leishmania infantum transmission cycle in wildlife, dogs and humans associated to vector abundance in Southeast Spain.

Leishmania infantum infection was investigated in 202 wild carnivores, rodents and lagomorphs in Southeast Spain using a real-time PCR (rtPCR) in skin and organ samples, mostly spleen. Lesions compatible with leishmaniosis were not observed in any of the animals. Prevalence defined as the percentage of rtPCR-positive animals was 32% overall, and 45% in foxes (n = 69), 30% in rabbits (n = 80) and stone martens (n = 10), 19% in wood mice (n = 16), 0% in black rats (n = 10) and ranged between 0% and 100% in other minoritarian species including badgers, wild cats, wolves, raccoons, genets and hares. Most infected rabbits were rtPCR-positive in skin and not in spleen samples and the opposite was the case for foxes (p < 0.05). L. infantum prevalence was lowest in spring following months of non-exposure to phlebotomine sand fly vectors, and spatially matched recently estimated Phlebotomus perniciosus vector abundance and the prevalence of subclinical infection in dogs and humans. Prevalence increased with altitude and was greater in drier and less windy South and West compared to the coastal Southeast of the study area (p < 0.05). Genetic diversity of L. infantum from foxes, investigated by PCR-restriction fragment length polymorphisms of kinetoplast DNA, revealed B genotype in all animals, which is frequent in people and dogs in the Iberian Peninsula and Morocco. The study provides further evidence that subclinical L. infantum infection is widespread in wildlife with prevalence depending on environmental factors and that parasite tissue tropism may vary according to host species. Moreover, it suggests that sylvatic and domestic transmission cycles are closely interconnected.

Anaphylaxis to diclofenac: nine cases reported to the Allergy Vigilance Network in France.

Nine cases of diclofenac hypersensitivity recorded by the Allergy Vigilance Network in France from 2002 to 2012 were studied. Data from history, symptoms, skin tests, basophil activation tests, and oral challenge (OC) were recorded. Grade 3 severe anaphylactic reactions occurred in seven cases of nine. IgE-dependent anaphylaxis was confirmed in six cases: positive intradermal tests (n = 4), a syndromic reaction during skin tests (n = 1), and one case with grade 1 reaction and negative skin tests had an anaphylactic shock to the OC. A nonimmune reaction was suspected in one case. An IgE-dependent mechanism may be the predominant cause of adverse reactions to diclofenac. Allergy skin tests must be carried out sequentially at the recommended concentrations. BATs may be helpful because they can support the diagnosis of anaphylaxis. Given the risks of a direct challenge to diclofenac, OC to aspirin should be performed first to exclude a nonimmunologic hypersensitivity to NSAIDs. Tests for specific IgEs to most frequently used NSAIDs such as diclofenac and ibuprofen are urgently needed.

Duocentric® reversed shoulder prosthesis and Personal Fit® templates: innovative strategies to optimize prosthesis positioning and prevent scapular notching.

We describe our experience with a new system of patient-specific template called Personal Fit(®), which is unique in shoulder surgery and used in combination with Duocentric(®) prosthesis. The reverse prosthesis's concept is the invention of Paul Grammont, developed with Grammont's team of Dijon University as from 1981, which led to the first reversed total shoulder prosthesis called Trumpet in 1985. The Duocentric(®) prosthesis developed in 2001 is the third-generation prosthesis, coming from the Trumpet and the second-generation prosthesis Delta(®) (DePuy). This prosthesis provides a novel solution to the notching problem with an inferior overhang integrated onto the glenoid baseplate. Personal Fit(®) system is based on reconstructing the shoulder joint bones in three dimensions using CT scan data, placing a landmark on the scapula and locating points on the glenoid and humerus. That will be used as a reference for the patient-specific templates. We study the glenoid position planned with Personal Fit(®) software relative to native glenoid position in 30 cases. On average, the difference between the planned retroversion (or anteversion in one case) and native retroversion was 8.6°.