Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells.

Bovine neosporosis is one of the major causes of reproductive failure in cattle worldwide, and differences in virulence between isolates have been widely shown. However, the molecular basis and mechanisms underlying virulence in Neospora caninum are mostly unknown. Recently, we demonstrated the involvement of NcGRA7 and NcROP40 in the virulence of N. caninum in a pregnant murine model using single knockout mutants in these genes generated by CRISR/Cas9 technology. In this study, the role of these proteins was investigated in two in vitro models using bovine target cells: trophoblast (F3 cell line) and monocyte-derived macrophages (BoMØ). The proliferation capacity of the single knockout mutant parasites was compared to the wild-type strain, the Nc-Spain7 isolate, using both cell populations. For the bovine trophoblast, no differences were observed in the growth of the defective parasites compared to the wild-type strain, neither in the proliferation kinetics nor in the competition assay. However, in naïve BoMØ, a significant decrease in the proliferation capacity of the mutant parasites was observed from 48 h pi onwards. Stimulation of BoMØ with IFN-γ showed a similar inhibition of tachyzoite growth in defective and wild-type strains in a dose-dependent manner. Finally, BoMØ infected with knockout parasites showed higher expression levels of TLR3, which is involved in pathogen recognition. These results suggest that NcGRA7 and NcROP40 may be involved in the manipulation of innate immune defense mechanisms against neosporosis and confirm the usefulness of the BoMØ model for the evaluation of N. caninum virulence mechanisms. However, the specific functions of these proteins remain unknown, opening the way for future research.

Enhancing Bioproducts in Seaweeds via Sustainable Aquaculture: Antioxidant and Sun-Protection Compounds.

Marine macroalgae are considered an untapped source of healthy natural metabolites and their market demand is rapidly increasing. Intertidal macroalgae present chemical defense mechanisms that enable them to thrive under changing environmental conditions. These intracellular chemicals include compounds that can be used for human benefit. The aim of this study was to test cultivation protocols that direct seaweed metabolic responses to enhance the production of target antioxidant and photoprotective biomaterials. We present an original integrated multi-trophic aquaculture (IMTA) design, based on a two-phase cultivation plan, in which three seaweed species were initially fed by fish effluents, and subsequently exposed to various abiotic stresses, namely, high irradiance, nutrient starvation, and high salinity. The combined effect of the IMTA's high nutrient concentrations and/or followed by the abiotic stressors enhanced the seaweeds' content of mycosporine-like amino acids (MAAs) by 2.3-fold, phenolic compounds by 1.4-fold, and their antioxidant capacity by 1.8-fold. The Sun Protection Factor (SPF) rose by 2.7-fold, and the chlorophyll and phycobiliprotein synthesis was stimulated dramatically by an order of magnitude. Our integrated cultivation system design offers a sustainable approach, with the potential to be adopted by emerging industries for food and health applications.

Neospora caninum infection induces an isolate virulence-dependent pro-inflammatory gene expression profile in bovine monocyte-derived macrophages.

BACKGROUND: Neospora caninum is an obligate intracellular parasite, and its ability to survive inside host immune cells may be a key mechanism for the establishment of infection in cattle. In vitro studies carried out by our group have shown that N. caninum is able to replicate in bovine macrophages (MØs), alter their microbicidal mechanisms and exploit their motility. Furthermore, host-cell control seems to be isolate virulence-dependent. METHODS: To investigate the molecular basis underlying the innate responses in MØs against N. caninum and the mechanisms of parasite manipulation of the host cell environment, the transcriptome profile of bovine monocyte-derived MØs infected with high-virulence (Nc-Spain7) or low-virulence (Nc-Spain1H) N. caninum isolates was studied. RESULTS: Functional enrichment revealed upregulation of genes involved in chemokine signalling, inflammation, cell survival, and inhibition of genes related with metabolism and phagolysosome formation. MØs activation was characterized by the induction of a predominantly M1 phenotype with expression of TLR2, TLR3 and TLR9 and activation of the NF-ƙB signalling pathway. Heat-killed N. caninum tachyzoites failed to activate NF-ƙB, and to inhibit lysosomal activity and apoptosis, which indicates active modulation by the parasite. The FoxO signalling pathway, Th1-Th2 differentiation, glycosaminoglycan degradation and apoptosis were pathways enriched only for low virulent Nc-Spain1H infection. In addition, Nc-Spain1H infection upregulated the IL12A and IL8 pro-inflammatory cytokines, whereas IL23 was downregulated by high virulent Nc-Spain7. CONCLUSIONS: This study revealed mechanisms implicated in the recognition of N. caninum by bovine MØs and in the development of the subsequent immune response. NF-ƙB seems to be the main signalling pathway implicated in the pro-inflammatory bovine MØs response against this pathogen. Apoptosis and phagolysosome maturation are processes repressed by N. caninum infection, which may guarantee its intracellular survival. The results also indicate that Nc-Spain7 may be able to partially circumvent the pro-inflammatory response whereas Nc-Spain1H induces a protective response to infection, which may explain the more efficient transmission of the high-virulence Nc-Spain7 isolate observed in vivo.

Crosstalk between Neospora caninum and the bovine host at the maternal-foetal interface determines the outcome of infection.

Neospora caninum is an apicomplexan cyst-forming parasite that is considered one of the main causes of abortion. The pathogenic mechanisms associated with parasite virulence at the maternal-foetal interface that are responsible for the outcome of infection are largely unknown. Here, utilizing placentomes from cattle experimentally infected with high-virulence (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates, we studied key elements of the innate and adaptive immune responses, as well as components of the extracellular matrix (ECM), at 10 and 20 days post-infection (dpi). The low-virulence isolate elicited a robust immune response characterized by upregulation of genes involved in pathogen recognition, chemokines and pro-inflammatory cytokines, crucial for its adequate control. In addition, Nc-Spain1H triggered the expression of anti-inflammatory cytokines and other mechanisms implicated in the maintenance of ECM integrity to ensure foetal survival. In contrast, local immune responses were initially (10 dpi) impaired by Nc-Spain7, allowing parasite multiplication. Subsequently (20 dpi), a predominantly pro-inflammatory Th1-based response and an increase in leucocyte infiltration were observed. Moreover, Nc-Spain7-infected placentomes from animals carrying non-viable foetuses exhibited higher expression of the IL-8, TNF-α, iNOS and SERP-1 genes and lower expression of the metalloproteases and their inhibitors than Nc-Spain7-infected placentomes from animals carrying viable foetuses. In addition, profound placental damage characterized by an alteration in the ECM organization in necrotic foci, which could contribute to foetal death, was found. Two different host-parasite interaction patterns were observed at the bovine placenta as representative examples of different evolutionary strategies used by this parasite for transmission to offspring.

Gene Expression Profiling of Neospora caninum in Bovine Macrophages Reveals Differences Between Isolates Associated With Key Parasite Functions.

Intraspecific differences in biological traits between Neospora caninum isolates have been widely described and associated with variations in virulence. However, the molecular basis underlying these differences has been poorly studied. We demonstrated previously that Nc-Spain7 and Nc-Spain1H, high- and low-virulence isolates, respectively, show different invasion, proliferation and survival capabilities in bovine macrophages (boMØs), a key cell in the immune response against Neospora, and modulate the cell immune response in different ways. Here, we demonstrate that these differences are related to specific tachyzoite gene expression profiles. Specifically, the low-virulence Nc-Spain1H isolate showed enhanced expression of genes encoding for surface antigens and genes related to the bradyzoite stage. Among the primary up-regulated genes in Nc-Spain7, genes involved in parasite growth and redox homeostasis are particularly noteworthy because of their correlation with the enhanced proliferation and survival rates of Nc-Spain7 in boMØs relative to Nc-Spain1H. Genes potentially implicated in induction of proinflammatory immune responses were found to be up-regulated in the low-virulence isolate, whereas the high-virulence isolate showed enhanced expression of genes that may be involved in immune evasion. These results represent a further step in understanding the parasite effector molecules that may be associated to virulence and thus to disease traits as abortion and transmission.

Differential Responses of Bovine Monocyte-Derived Macrophages to Infection by Neospora caninum Isolates of High and Low Virulence.

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, represents one of the main causes of abortion in cattle. Macrophages (MØs) are mediators of the innate immune response against infection and likely one of the first cells encountered by the parasite during the host infection process. In this study, we investigated in vitro how high or low virulent isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively) interact with bovine monocyte-derived MØs and the influence of the isolate virulence on the subsequent cellular response. Both isolates actively invaded, survived and replicated in the MØs. However, Nc-Spain7 showed a higher invasion rate and a replication significantly faster, following an exponential growth model, whereas Nc-Spain1H presented a delayed replication and a lower growth rate without an exponential pattern. N. caninum infection induced a hypermigratory phenotype in bovine MØs that was characterized by enhanced motility and transmigration in vitro and was accompanied by morphological changes and abrogated extracellular matrix degradation. A significantly higher hypermotility was observed with the highly virulent isolate Nc-Spain7. Nc-Spain1H-infected MØs showed elevated reactive oxygen species (ROS) production and IL12p40 expression, which also resulted in increased IFN-γ release by lymphocytes, compared to cells infected with Nc-Spain7. Furthermore, IL-10 was upregulated in MØs infected with both isolates. Infected MØs exhibited lower expression of MHC Class II, CD86, and CD1b molecules than uninfected MØs, with non-significant differences between isolates. This work characterizes for the first time N. caninum replication in bovine monocyte-derived MØs and details isolate-dependent differences in host cell responses to the parasite.

Dual prophylaxis with teicoplanin and cefazolin in the prevention of prosthetic joint infection. / Profilaxis dual con teicoplanina añadida a cefazolina en la prevención de la infección de prótesis articular.

INTRODUCTION: There is a growing increase in prosthetic joint infection (PJI) incidence due to cephalosporin-resistant bacteria, used in surgical prophylaxis. The replacement of these with glycopeptides has not been shown to improve the results, but they have been shown to improve with their combination. METHODS: Comparative study of combination of teicoplanin and cefazolin before arthroplasty surgery against cefazolin alone from a previous control group. RESULTS: During the control period, there were 16 PJIs from 585 surgeries, while in the intervention group there were 6 from 579 (incidence 2.7% vs. 1.03%, RR 0.4, P=.04). In control group, 11 of the infections were caused by Gram-positive bacteria versus 4 in the intervention group (1.8% vs. 0.7%, P=.08). CONCLUSIONS: The addition of teicoplanin to cefazolin in the prophylaxis of arthroplasty surgery was associated with a reduction in the incidence of PJI, thanks to a decrease in infections caused by Gram-positive bacteria.

Ultrasound-guided Ex-vivo Retrieval of Mature Oocytes for Fertility Preservation During Laparoscopic Oophorectomy: A Case Report.

BACKGROUND: Cryopreservation of oocytes is an efficient method of fertility preservation (FP) that can be applied in women suffering from gynecologic conditions that menace their reproductive future. Collection of oocytes becomes challenging in some scenarios, like the possibility of an ovarian cancer, the "ex-vivo" harvest of oocytes for FP, aspirating follicles directly from the ovarian specimen already excised by laparotomy or laparoscopy and it is an option for these cases. CASE PRESENTATION: In the present case report, the case of a patient with an adnexal mass suspected to be a recurrent teratoma was described who referred to our Assisted Reproduction Unit in Hospital Quironsalud Malaga for FP counseling. After controlled ovarian hyperstimulation, followed by laparoscopic abdominal examination and oophorectomy, an ex-vivo follicular aspiration for oocyte retrieval was performed on the specimen, using a standard ultrasound-guided procedure to ease and improve the process. All the follicles were aspirated and 5 metaphase II oocytes were obtained. CONCLUSION: This is to our knowledge, the first communication describing the ex-vivo ovarian aspiration of mature oocytes for FP using standard ultrasound guidance. Although this ultrasound guidance is not completely necessary, as other authors demonstrated previously, such a procedure permitted an easy and complete harvest of oocytes in a rare tumor with bizarre cystic formations, which made follicle recognition very difficult.

Iron supplementation in mouse expands cellular innate defences in spleen and defers lethal malaria infection.

The co-endemicity of malnutrition, erythrocytopathies, transmissible diseases and iron-deficiency contribute to the prevalence of chronic anaemia in many populations of the developing world. Although iron dietary supplementation is applied or recommended in at risk populations, its use is controversial due to undesirable outcomes, particularly regarding the response to infections, including highly prevalent malaria. We hypothesized that a boosted oxidative stress due to iron supplementation have a similar impact on malaria to that of hereditary anaemias, enhancing innate response and conditioning tissues to prevent damage during infection. Thus, we have analysed antioxidant and innate responses against lethal Plasmodium yoelii during the first five days of infection in an iron-supplemented mouse. This murine model showed high iron concentration in plasma with upregulated expression of hemoxygenase-1. The sustained homeostasis after this extrinsic iron conditioning, delayed parasitemia growth that, once installed, developed without anaemia. This protection was not conferred by the intrinsic iron overload of hereditary hemochromatosis. Upon iron-supplementation, a large increase of the macrophages/dendritic cells ratio and the antigen presenting cells was observed in the mouse spleen, independently of malaria infection. Complementary, malaria promoted the splenic B and T CD4 cells activation. Our results show that the iron supplementation in mice prepares host tissues for oxidative-stress and induces unspecific cellular immune responses, which could be seen as an advantage to promote early defences against malaria infection.

DoSurgeons Have More Difficulties in the Hospital Care of Non-surgery Patients Than With Surgery Patients?

OBJECTIVE: A variable percentage of patients admitted to surgical departments are not operated on for several reasons. Our goal is to check if surgeons have more problems in caring for non-operated hospitalized patients than operated ones. MATERIAL AND METHODS: We included all patients aged ≥ 14 years discharged in 2010 from General Surgery, Gynaecology, Urology, and Otolaryngology. The main variables were the length of stay, mortality, readmissions, and number of consultations/referrals requested to medical services. Secondary variables were age, sex, number of emergency admissions, total number of diagnoses, and the Charlson comorbidity index (ICh). RESULTS: Between 8.7% and 22.8% of patients admitted to these surgical departments are not operated on. The non-operated patients had a significantly higher stay, mortality, readmissions and consultations/referrals requests than operated ones, with significantly higher age (except Urology), number of diagnoses, emergency admissions and ICh (except Urology). CONCLUSIONS: Patients admitted to surgical departments and are not operated on have higher mortality, readmissions and consultation/referrals requests than those operated on, which may be due to their greater medical complexity and urgency of admission. This suggests a greater difficulty in their care by surgeons.

Living in a coastal lagoon environment: photosynthetic and biochemical mechanisms of key marine macroalgae.

The physiological status of Cystoseira compressa, Padina pavonica and Palisada tenerrima was studied by in vivo chlorophyll fluorescence, pigment content, stoichiometry (C:N), accumulation of UV photoprotectors and antioxidant activity; comparing their photosynthetic response in a coastal lagoon (Mar Menor) and in Mediterranean coastal waters. In general, the specimens reached their highest ETRmax in spring in the Lagoon, but in summer in the Mediterranean, coinciding with their maximum biomass peak. The species exhibited a dynamic photoinhibition. Except C. compressa, they showed a lower decrease in Fv/Fm and higher recovery rates in the Mediterranean populations when exposed to high irradiance. The higher salinity and temperature of the lagoon could impair the photoprotection mechanisms. The acclimation to lagoon environments is species-specific and involves complex regulatory mechanisms. The results underline the importance of N in repair, avoidance, quenching and scavenging mechanisms. In general, Lagoon specimens showed higher pigment concentration. Although xanthophylls play important photo-protective and antioxidant roles, the observed trend is more likely to be explained by the higher temperatures reached in the lagoon compared to Mediterranean. Therefore the studied photosynthetic and biochemical mechanisms can be effective not only for high irradiance, but also for higher temperatures in a climate change scenario, but are highly dependent on nutrient availability.

Intervención educativa para la promoción de la lactancia materna / Educational intervention for the promotion of maternal lactancy

Introducción: la lactancia materna es un hecho biológico sujeto a modificaciones por influencias sociales, económicas y culturales, que ha permanecido como referente para los bebés humanos desde los orígenes de la humanidad hasta finales del siglo XIX.Objetivo: evaluar el efecto de un programa de intervención educativa en el conocimiento que sobre lactancia materna tienen las embarazadas que residen en el área del Policlínico Universitario Marta Martínez del municipio Güines, que en el periodo comprendido desde julio hasta diciembre del 2012.Métodos: se realizó un estudio cuasi-experimental, específico de antes y después. La muestra estuvo conformada por un total de 84 gestantes que se encontraban en el tercer trimestre del embarazo y residían en el área urbana. Se aplicó una encuesta para conocer el nivel de conocimiento que existía sobre el tema, se aplicó un programa educativo y se ofreció un folleto ilustrativo e instructivo sobre la lactancia materna, se aplicó nuevamente el cuestionario para explorar los conocimientos adquiridos.Resultados: más de la mitad de las embarazadas tenían un nivel de conocimiento antes de la intervención de regular resultados que se revirtieron al concluir la misma. La aplicación de programa educativo permitió el aprendizaje y la ejecución correcta del proceder en la mayor parte de las integrantes del grupo. Conclusiones: el conocimiento aún es insuficiente en las embarazadas y se demuestra la efectividad de las técnicas educativas, por lo que la actividad de promoción ya sea a través de charlas educativas, folletos ilustrados o individualmente es necesaria en las gestantes

Introduction: maternal lactancy is a biological fact subject to modifications by social, economic and cultural influences, which has remained as a reference for human babies since the beginning of mankind until the late nineteenth century.Objective: to assess the effect of an educational intervention program on the knowledge about maternal lactancy that pregnants residing in the area of Marta Martínez University Polyclinic of Güines municipality have, in the period from July to December 2012.Methods: a specific before and after quasi-experimental study was performed. The sample consisted of a total of 84 pregnant women who were in the third trimester and residing in the urban area. A survey to assess the level of knowledge that existed on the subject was applied, an educational program was implemented and an illustrative and instructive booklet on maternal lactancy was offered, the questionnaire was applied again to explore the knowledge acquired.Results: more than half of the pregnant women had a level of understanding before the intervention of regular results which reverted when concluding it. The implementation of the educational program enabled learning and proper execution of the procedure in most of the group members.Conclusions: knowledge is still insufficient in pregnants and the effectiveness of educational techniques are demonstrated, so the promotion activity either through educational talks, illustrated brochures or individually is necessary in pregnant women

[Factors that influence interdepartmental referrals between Surgical Departments and Internal Medicine]. / Factores que influyen en la solicitud de interconsultas a medicina interna por los servicios quirúrgicos.

INTRODUCTION: To analyse the long term outcome of the age and comorbidity of patients admitted to Surgical Departments, the number of referrals to Internal Medicine made by these Departments, and to assess whether there are seasonal variations and the call/reject effect. MATERIAL AND METHODS: We compared the age, Charlson Comorbidity Index (CCI), and the number of referrals made by Traumatology, General Surgery and Urology of patients discharged in 2000, with those discharged in 2007. Seasonal variations and the call/reject effect were studied by analysing all the interdepartmental referrals made by all the surgical departments from the year 2000 to 2007. RESULTS: Age increased by 5.6% between 2000 and 2007, the CCI by 5.8%, and interdepartmental referrals by 60%. Interdepartmental referrals decreased in July and August, whilst they increased in January, February, June and October, up to 64% more in January, although with variations of almost 50% in the same month. We detected differences of up to 68.2% in the referrals requested to different physicians. CONCLUSIONS: We observed a sharp increase in the requests for referral to Internal Medicine by Surgical Departments of our hospital, which is not explained by the increase in admissions to these Departments, and which could be associated with the increase in age and comorbidity of their patients. Requests for interdepartmental referral have marked monthly variations and also as regards the Consulting Physician.