Carolina Hurtado

The Viral Protein A238L Inhibits TNF-α Expression through a CBP/p300 Transcriptional Coactivators Pathway

African swine fever virus (ASFV) is able to inhibit TNF-α-induced gene expression through the synthesis of A238L protein. This was shown by the use of deletion mutants lacking the A238L gene from the Vero cell-adapted Ba71V ASFV strain and from the virulent isolate E70. To further analyze the molecular mechanism by which the viral gene controls TNF-α, we have used Jurkat cells stably transfected with the viral gene to identify the TNF-α regulatory elements involved in the induction of the gene after stimulation with PMA and calcium ionophore. We have thus identified the cAMP-responsive element and κ3 sites on the TNF-α promoter as the responsible of the gene activation, and demonstrate that A238L inhibits TNF-α expression through these DNA binding sites. This inhibition was partially reverted by overexpression of the transcriptional factors NF-AT, NF-κB, and c-Jun. Furthermore, we present evidence that A238L inhibits the activation of TNF-α by modulating NF-κB, NF-AT, and c-Jun trans activation through a mechanism that involves CREB binding protein/p300 function, because overexpression of these transcriptional coactivators recovers TNF-α promoter activity. In addition, we show that A238L is a nuclear protein that binds to the cyclic AMP-responsive element/κ3 complex, thus displacing the CREB binding protein/p300 coactivators. Taken together, these results establish a novel mechanism in the control of TNF-α gene expression by a viral protein that could represent an efficient strategy used by ASFV to evade the innate immune response.

The viral protein A238L inhibits cyclooxygenase-2 expression through a nuclear factor of activated T cell-dependent transactivation pathway

Cyclooxygenase-2 is transiently induced upon cell activation or viral infections, resulting in inflammation and modulation of the immune response. Here we report that A238L, an African swine fever virus protein, efficiently inhibits cyclooxygenase-2 gene expression in Jurkat T cells and in virus-infected Vero cells. Transfection of Jurkat cells stably expressing A238L with cyclooxygenase-2 promoter-luciferase constructs containing 5′-terminal deletions or mutations in distal or proximal nuclear factor of activated T cell (NFAT) response elements revealed that these sequences are involved in the inhibition induced by A238L. Overexpression of a constitutively active version of the calcium-dependent phosphatase calcineurin or NFAT reversed the inhibition mediated by A238L on cyclooxygenase-2 promoter activation, whereas overexpression of p65 NFκB had no effect. A238L does not modify the nuclear localization of NFAT after phorbol 12-myristate 13-acetate/calcium ionophore stimulation. Moreover, we show that the mechanism by which the viral protein down-regulates cyclooxygenase-2 activity does not involve inhibition of the binding between NFAT and its specific DNA sequences into the cyclooxygenase-2 promoter. Strikingly, A238L dramatically inhibited the transactivation mediated by a GAL4-NFAT fusion protein containing the N-terminal transactivation domain of NFAT1. Taken together, these data indicate that A238L down-regulates cyclooxygenase-2 transcription through the NFAT response elements, being NFAT-dependent transactivation implicated in this down-regulation.

The use of COS-1 cells for studies of field and laboratory African swine fever virus samples.

Different naturally occurring, cell adapted or genetically manipulated stocks of African swine fever virus were able to infect directly cultures of COS-1 cells, producing extensive cytopathic effects and amounts from 10(6) to 10(7) of infective progeny virus per ml. The induction of late virus-specific proteins, demonstrated by RT-PCR and immunoblotting, and the development of lysis plaques by all the virus samples tested so far, allowed the optimization of both titration and diagnostic assays, as well as the proposal of a method for selection of virus clones during the generation of virus mutants with specific gene deletions.

First Case of Legionnaire's Disease Caused by Legionella anisa in Spain and the Limitations on the Diagnosis of Legionella non-pneumophila Infections.

Legionnaires’ disease is a severe form of pneumonia, with worldwide relevance, caused by Legionella spp. Approximately 90% of all cases of legionellosis are caused by Legionella pneumophila, but other species can also be responsible for this infection. These bacteria are transmitted by inhalation of aerosols or aspiration of contaminated water. In Spain, environmental studies have demonstrated the presence of Legionella non-pneumophila species in drinking water treatment plants and water distribution networks. Aware that this evidence indicates a risk factor and the lack of routine assays designed to detect simultaneously diverse Legionella species, we analyzed 210 urine samples from patients presenting clinical manifestations of pneumonia using a semi-nested PCR for partial amplification of the 16S rDNA gene of Legionella and a diagnostic method used in hospitals for Legionella antigen detection. In this study, we detected a total of 15 cases of legionellosis (7.1%) and the first case of Legionnaires’ disease caused by L. anisa in Spain. While the conventional method used in hospitals could only detect four cases (1.9%) produced by L. pneumophila serogroup 1, using PCR, the following species were identified: Legionella spp. (10/15), L. pneumophila (4/15) and L. anisa (1/15). These results suggest the need to change hospital diagnostic strategies regarding the identification of Legionella species associated with this disease. Therefore, the detection of Legionella DNA by PCR in urine samples seems to be a suitable alternative method for a sensitive, accurate and rapid diagnosis of Legionella pneumonia, caused by L. pneumophila and also for L. non-pneumophila species.

Acanthamoeba spp. in Contact Lenses from Healthy Individuals from Madrid, Spain

Purpose Acanthamoeba keratitis (AK) is a painful and potentially blinding corneal infection caused by Acanthamoeba spp. In Madrid, environmental studies have demonstrated a high presence of these free-living amoebae in tap water. Since most of AK cases occur in contact lenses (CL) wearers with inadequate hygiene habits, the presence of Acanthamoeba in discarded CL has been studied and compared with other common etiological agents of keratitis, such as Pseudomonas aeruginosa and Staphylococcus aureus. Methods One hundred and seventy-seven healthy individuals from Madrid contributed their discarded CL and answered a questionnaire on hygiene habits. DNA was extracted from the CL solution and analyzed by real-time PCR for Acanthamoeba, Pseudomonas aeruginosa and Staphylococcus aureus. These CL and their solutions were also cultured on non-nutrient agar to isolate Acanthamoeba. Results Among the 177 samples, Acanthamoeba DNA was detected in 87 (49.2%), P. aeruginosa DNA in 14 (7.9%) and S. aureus DNA in 19 (10.7%). Cultivable amoebae, however, were observed in only one sample (0.6%). This isolate was genotyped as T4. The habits reported by this CL owner included some recognized risk factors for AK, but in this study only the practice of “not cleaning the CL case” presented some statistical significant association with Acanthamoeba DNA presence. Detection of the investigated bacterial DNA did not demonstrate statistical significant association with the studied practices, but the presence of P. aeruginosa revealed a possible inhibition of Acanthamoeba in these samples. Conclusions The PCR results suggest a high presence of Acanthamoeba spp. in healthy CL wearers from Madrid, but we can assume that CL solutions are properly disinfecting the CL since only 1.1% of the positive PCR samples correspond to viable amoebae and, after four years, only one participant reported stronger ocular problems. Nevertheless, more studies are necessary to corroborate this hypothesis.

Development of a virtual environment for teaching and learning biomedical techniques and equipment for the study of human pathogens.

An international innovative teaching group from different EU Universities (De Montfort University, Leicester, UK; University of San Pablo CEU, Madrid, Spain; University of Miguel Hernandez, Elche, Spain) and biomedical scientists registered by the Health and Care Professions Council (HCPC, UK) are developing a complete e-learning package in medical parasitology for undergraduate and postgraduate students that study Health Sciences. This package, named DMU e-Parasitology, is accessible through the DMU website (http://parasitology.dmu.ac.uk) and will present different modules including a virtual laboratory module for the study of traditional and novel biomedical laboratory techniques and equipment for detecting, identifying and studying human pathogens, specifically parasites. These techniques could also be potentially used to study other pathogens such as bacteria or viruses. The virtual biomedical laboratory is under development, but is available in the DMU website here: http://parasitology.dmu.ac.uk/learn/laboratory.htm. To develop this new module of the DMU eParasitology, we are using Storyline 360 software and the scaffolding and methods used to build the theoretical module (Peña-Fernández et al., 2017) [1]. To facilitate the navigation, study and comprehension of the final user, we have divided the virtual laboratory into a series of sub-sections that include different units; the sub-sections so far are: microscopes (with units such as the electron microscope); molecular biology (e.g. polymerase chain reaction and gel electrophoresis); biological safety cabinets and cell/parasite culture; biochemical and immunological techniques (e.g. magnetic immunoseparation); histology (e.g. microtome) and staining techniques (e.g. Kinyoun staining). The virtual laboratory units are highly interactive and present short videos of academics and/or technicians working in real conditions with the different laboratory equipment such as a thermocycler, a microtome, or a biological safety cabinet, as well as performing a specific technique such as a staining to determine pathogens. Therefore, the user of this virtual environment will receive a complete and “real” experience of the work in a biomedical laboratory. The DMU e-Parasitology package, and specifically its virtual laboratory environment, could help technicians and students across the world to learn how to work in a biomedical laboratory as well as to perform techniques to identify and diagnose human pathogens such as microsporidia or Plasmodium spp. Thus, the virtual resource is supported by a virtual library that includes a real collection of clinical slides that will provide the user with the functionality of a light and/or an immunofluorescence microscope. In conclusion, the virtual laboratory may serve as a high quality and reliable on-line environment for the learning of techniques and equipment. These resources can be used to improve the learning of undergraduate and postgraduate students of human health sciences as well as to develop CPD training. Moreover, the virtual laboratory module may impact in the teaching of laboratory techniques and skills in developing countries due to their limited resources. This communication will explore the design and development of the virtual laboratory environment that will be publicly accessible by the end of 2018.

Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp . spores and their developmental stages

BackgroundMicrosporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult and immunological techniques such as immunofluorescence have proved to be a useful and reliable tool in the diagnosis of human microsporidiosis. For this reason, we have produced and characterized monoclonal antibodies (MAbs) specific for Encephalitozoon intestinalis (the second most frequent microsporidian infecting humans), and other Encephalitozoon species, that can be used in different diagnostic techniques.ResultsSeven MAbs were selected in accordance with their optical density (OD). Four (4C4, 2C2, 2E5 and 2H2) were isotype IgG2a; two (3A5 and 3C9) isotype IgG3, and one Mab, 1D7, IgM isotype. The selected monoclonal antibody-secreting hybridomas were characterized by indirect immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), Western blot, immunoelectron microscopy (Immunogold) and in vitro cultures. The study by IFAT showed different behavior depending on the MAbs studied. The MAbs 4C4, 2C2, 2E5 and 2H2 showed reactivity against epitopes in the wall of the spore (exospore and endospore) epitopes located in Encephalitozoon sp. spores, whereas the MAbs 3A5, 1D7 and 3C9 showed reactivity against internal epitopes (cytoplasmic contents or sporoplasm) of E. intestinalis spores. All MAbs recognized the developing parasites in the in vitro cultures of E. intestinalis. Additionally, 59 formalin-fixed stool samples that had been previously analyzed were screened, with 26 (44%) presenting microsporidian spores (18 samples with E. intestinalis and 8 samples with Enterocytozoon bieneusi). Detection of microsporidian spores by microscopy was performed using Calcofluor stain, Modified Trichrome, Quick-Hot Gram Chromotrope, as well as IFAT using MAbs 4C4, 2C2, 2E5 and 2H2. The 4 MAbs tested clearly recognized the larger spores corresponding to E. intestinalis, but showed no reactivity with Enterocytozoon bieneusi spores. The mass spectrometry and proteomic study revealed that the Mabs 4C4, 2C2, 2E5 and 2H2 recognized the Spore Wall Protein 1 (SWP1) as the antigenic target.ConclusionsThe IFAT-positive MAbs exhibited excellent reactivity against spores and developmental stages, permitting their use in human and animal diagnosis. The epitopes recognized (exospore, endospore and cytoplasmic contents) by the different MAbs developed need further study, and may reveal potential targets for vaccine development, immunotherapy and chemotherapy.

Environmental recovery training for biological incidents: impressions from pharmacy students

An innovative teaching group at De Montfort University (DMU, UK) and at the University of Alcalá (Spain) has developed specific training to prepare human health students to respond to biological incidents. The purpose of this training is to provide a basic understanding of environmental toxicology, recovery, public health and medical preparedness to protect humans and minimise the spread of biological hazards. We have followed previous experience gained when responding to the 2014-16 Ebola outbreak in Makeni, Sierra Leone (West Africa). Previously, to create this training, a series of basic competences to respond to biological incidents were created by our group for undergraduate students [1] following the European Commission competences specified for medical responders [2]. A critical part of any intervention plan to respond to these incidents is to implement a quick response to protect the public and actions to minimise the occurrence of infections and recover the environment affected by the biological agent(s) since they can subsequently impact humans. A specific short training course/workshop (3 hours long) has been created in conjunction with a series of specific lectures on emerging pathogens, biological hazards and prevention, medical preparedness and public health. Students were provided with a biological incident scenario affecting different environments (open water and food production systems) and they needed to tailor a basic recovery strategy following the novel methodology and guidelines to tailor a recovery response to biological incidents developed by Public Health England (PHE) [3]. Our recovery training has been tested within different undergraduate and postgraduate programmes (BSc Biomedical and BMedSci Medical Science; MSc Advances in Biomedical Science) at DMU during the 2016/17 academic course; we have determined high levels of student satisfaction and engagement. Currently we are in the process of validating this training by delivering it in other European universities and/or different health care programmes. Thus, we delivered a modified version of this recovery training/workshop to third year pharmacy students (n=101) enrolled in the module Biological Analysis & Laboratory Diagnostic at the Universidad de San Pablo CEU (Spain) in April 2017. Pharmacy students gain an appropriate knowledge on infectious diseases, microbiology and parasitology during the first two years of study, so we decided to reduce the length of the workshop to 1.5 hours. A validated feedback-questionnaire was distributed that has revealed the following initial impressions (33 students completed the questionnaire): 93.9% of students enjoyed the different exercises created (45.4% agreed; 48.5% strongly agreed); only 6.1% of them did not (6.1% disagreed; 0% strongly disagreed). 87.9% of these students reported that the novel PHE resources used aided their learning on environmental recovery and restoration. Finally, 93.9 % of students (42.4% agreed; 51.5% strongly agreed) indicated that they have gained knowledge to tailor a basic recovery plan in the aftermath of a biological incident; 6.1% disagreed. These results should be considered as preliminary and the training was shown to be successful in facilitating health care students to acquire basic skills to recover environments affected by future biological incidents and outbreaks of infection.