Murine Parainfluenza Virus Persists in Lung Innate Immune Cells Sustaining Chronic Lung Pathology.

Respiratory viruses including the human parainfluenza viruses (hPIVs) are a constant burden to human health, with morbidity and mortality frequently increased after the acute phase of the infection. Although is proven that respiratory viruses can persist in vitro, the mechanisms of virus or viral products persistence, their sources, and their impact on chronic respiratory diseases in vivo are unknown. Here, we used Sendai virus (SeV) to model hPIV infection in mice and test whether virus persistence associates with the development of chronic lung disease. Following SeV infection, virus products were detected in lung macrophages, type 2 innate lymphoid cells (ILC2s) and dendritic cells for several weeks after the infectious virus was cleared. Cells containing viral protein showed strong upregulation of antiviral and type 2 inflammation-related genes that associate with the development of chronic post-viral lung diseases, including asthma. Lineage tracing of infected cells or cells derived from infected cells suggests that distinct functional groups of cells contribute to the chronic pathology. Importantly, targeted ablation of infected cells or those derived from infected cells significantly ameliorated chronic lung disease. Overall, we identified persistent infection of innate immune cells as a critical factor in the progression from acute to chronic post viral respiratory disease.

Non-standard viral genome-derived RNA activates TLR3 and type I IFN signaling to induce cDC1-dependent CD8+ T-cell responses during vaccination in mice.

There is a critical need to develop vaccine adjuvants that induce robust immune responses able to protect against intracellular pathogens, including viruses. Previously, we described defective viral genome-derived oligonucleotides (DDOs) as novel adjuvants that strongly induce type 1 immune responses, including protective Th1 CD4+ T-cells and effector CD8+ T-cells in mice. Here, we unravel the early innate response required for this type 1 immunity induction. Upon DDO subcutaneous injection, type 1 conventional dendritic cells (cDC1s) accumulate rapidly in the draining lymph node in a Toll-like receptor 3 (TLR3)- and type I interferon (IFN)-dependent manner. cDC1 accumulation in the lymph node is required for antigen-specific CD8+ T-cell responses. Notably, in contrast to poly I:C, DDO administration resulted in type I IFN expression at the injection site, but not in the draining lymph node. Additionally, DDOs induced an inflammatory cytokine profile distinct from that induced by poly I:C. Therefore, DDOs represent a powerful new adjuvant to be used during vaccination against intracellular pathogens.

A New Cage-Like Particle Adjuvant Enhances Protection of Foot-and-Mouth Disease Vaccine.

Foot-and-Mouth Disease (FMD) is an acute viral disease that causes important economy losses. Vaccines with new low-cost adjuvants that stimulate protective immune responses are needed and can be assayed in a mouse model to predict their effectiveness in cattle. Immunostimulant Particle Adjuvant (ISPA), also known as cage-like particle adjuvant, consisting of lipid boxes of dipalmitoyl-phosphatidylcholine, cholesterol, sterylamine, alpha-tocopherol, and QuilA saponin, was shown to enhance protection of a recombinant vaccine against Trypanosoma cruzi in a mouse model. Thus, in the present work, we studied the effects on the magnitude and type of immunity elicited in mice and cattle in response to a vaccine based on inactivated FMD virus (iFMDV) formulated with ISPA. It was demonstrated that iFMDV-ISPA induced protection in mice against challenge and elicited a specific antibody response in sera, characterized by a balanced Th1/Th2 profile. In cattle, the antibody titers reached corresponded to an expected percentage of protection (EPP) higher than 80%. EPP calculates the probability that livestock would be protected against a 10,000 bovine infectious doses challenge after vaccination. Moreover, in comparison with the non-adjuvanted iFMDV vaccine, iFMDV-ISPA elicited an increased specific T-cell response against the virus, including higher interferon gamma (IFNγ)+/CD8+ lymphocyte production in cattle. In this work, we report for first time that an inactivated FMDV serotype A vaccine adjuvanted with ISPA is capable of inducing protection against challenge in a murine model and of improving the specific immune responses against the virus in cattle.

Mouse model as an efficacy test for foot-and-mouth disease vaccines.

Protection against foot-and-mouth disease virus (FMDV) has been linked to the development of a humoral response. In Argentina, the official control tests for assessing the potency of FMD vaccines are protection against podal generalization (PPG) and expected percentage of protection (EPP) curves built with quantitative data of antibodies determined by liquid-phase blocking ELISA (lpELISA). The results of these tests are used to accept or discard vaccines at the batch level. In this report, a mouse model was assessed as an alternative efficacy control for FMDV vaccines. To this aim, groups of cattle (n = 18) and BALB/c mice (n = 16) were inoculated with commercial FMDV vaccines and bleedings were performed 60 days post vaccination (dpv) in cattle and 21 dpv in mice. Specific FMDV antibody titres were measured in both species by a standardized lpELISA. A statistically significant association between antibody levels in cattle and mice has already been demonstrated. However, some vaccines have been misclassified since they were considered protective based on lpELISA results but did not induce good protection in cattle upon challenge. For this reason, other immunological parameters were evaluated to improve the prediction of protection in mice, without the need of using infective virus. In addition, antibody titres by lpELISA, the IgG2b/IgG1 isotype ratio and the Avidity Index were identified as good predictors, resulting in an optimal predictive model of protection. This mouse model could be a simple and economic alternative for testing FMD vaccines since the disadvantages of high costs and facility requirements associated with the use of large animals are overcome.

Peces de consumo humano como indicadores de contaminación ambiental por plaguicidas en el norte de Misiones, Argentina / Fishes for human consumption as indicators of environmental pollution by pesticides in northern Misiones, Argentina

RESUMEN INTRODUCCIÓN El norte de la provincia de Misiones se caracteriza por una gran superficie de cultivos en pequeña escala, que es necesario investigar debido a su impacto socioambiental por el uso de plaguicidas. Además, la acuicultura en esta zona del país se ha expandido en los últimos años con la introducción de programas familiares como mecanismo de diversificación de ingresos. Mediante la utilización de peces de consumo humano como indicadores de contaminación, este proyecto tuvo por objetivo dar una primera aproximación a los riesgos ambientales y sanitarios surgidos como consecuencia de prácticas de manejo inadecuadas en el uso multifuncional de la tierra. MÉTODOS Se realizó el análisis de biomarcadores no destructivos en peces de la especie Oreochromis niloticus, como el recuento diferencial de leucocitos, la actividad enzimática de la colinesterasa y la frecuencia de micronúcleos. RESULTADOS Se determinaron alteraciones en los valores de estos parámetros en individuos pertenecientes a la zona de cultivos con respecto a los individuos de áreas protegidas, y se observó la presencia de lindano en el sedimento de los cuerpos de agua de la zona de cultivo. CONCLUSIONES Aunque el manejo multifuncional de la tierra en este área contribuyó a un uso más eficiente de los recursos, la exposición de los peces a los pesticidas utilizados en los cultivos se ha convertido en una consecuencia involuntaria en la región.

ABSTRACT INTRODUCTION The northern region of Misiones is characterized by a large area of small-scale crops, whose socio-environmental impact through use of pesticides has to be studied. The combination of crops and aquaculture in this region grew significantly as an income diversification mechanism. Through the use of fish for human consumption as indicator of pollution, this project aimed to determine environmental and health risks associated to inadequate land-use management practices. METHODS Analyses of non-destructive biomarkers like differential leukocyte count, cholinesterase enzymatic activity and frequency of micronuclei in fish of the species Oreochromis niloticus were carried out. RESULTS Alterations of the standard parameters tested on individuals from crop areas were determined and the level of lindane in sediment of the ponds in culture zone was detected. CONCLUSIONS Although the multi-functional land management led to a more efficient use of natural resources, the exposure of fish to pesticides used on the crops has become an unintended consequence in this region.

Peces de consumo humano como indicadores de contaminación ambiental / Fish for human consumption as indicators of environmental pollution

El norte de la provincia de Misiones se caracteriza por poseer una gran superficie de producción de cultivos de pequeña escala, los cuales por su impacto socio ambiental, debido al uso de plaguicidas, se encuentran fuertemente observados. Además, la acuicultura en esta zona del país se ha estado expandiendo en los ultimos años debido a la introduccion de programas de acuicultura familiar como un mecanismo de diversificacion de ingresos. Este proyecto, mediante la utilización de peces de consumo humano como indicadores de la contaminación, busca determinar los riesgos ambientales y sanitarios consecuencia de prácticas de manejo inadecuadas en el uso multifuncional de la tierra. A partir del análisis de biomarcadores hematológicos como el recuento diferencial de leucocitos, el análisis de micronúcleos y actividad enzimática colinesterasa en peces de la especie Oreochromis niloticus se determinaron alteraciones en los parámetros normales analizados en individuos pertenecientes a zonas de cultivos. Aunque el uso multifuncional de la tierra en esta área ha llevado a un uso más eficiente de los recursos, la exposición de los peces a los pesticidas utilizados en los cultivos se ha convertido en una consecuencia involuntaria común en esta región

Sensibilidad gustativa en Apis mellifera como indicador de la contaminación ambiental en Misiones / Taste sensitivity in Apis mellifera as an indicator of environmental contamination in Misiones

La producción de tabaco requiere la utilización de una amplia variedad de agrotóxicos. El uso inadecuado de insecticidas y herbicidas puede provocar por un lado la contaminación de cursos de agua y por otro, la acumulación en animales y humanos, generando graves problemas de salud como intoxicación y deficiencias en el desarrollo. Las abejas Apis mellifera pueden actuar como excelentes indicadores biológicos de la contaminación del ambiente por ser importantes polinizadores de la flora nativa y cultivos. En las abejas que visiten las flores de cultivos tratados con agrotóxicos y flora lindante, se verán afectadas las capacidades cognitivas y de comportamiento utilizando estos cambios como indicadores de contaminación ambiental. En este trabajo se utilizó como variable a medir, la sensibilidad gustativa a la sacarosa en las abejas Apis mellifera, la cual se basa en registrar la respuesta de extensión de probóscide (REP) de las abejas para una serie de concentraciones crecientes de sacarosa y obtener de esta manera los umbrales de respuesta al azúcar. Las abejas provenientes de colmenas ubicadas en áreas afectadas por los agrotóxicos, mostraron mayores umbrales (menor sensibilidad) de sacarosa que aquellas abejas que viven y recolectan de flores de zonas libres de agrotóxicos. En este trabajo se pudo obtener una primera aproximación de la calidad ambiental de las áreas afectadas por los plaguicidas del norte de la provincia de Misiones

A DNA Vaccine Formulated with Chemical Adjuvant Provides Partial Protection against Bovine Herpes Virus Infection in Cattle.

Bovine herpesvirus-1 (BoHV-1) is the causative agent of bovine infectious rhinotracheitis, an important disease worldwide. Although conventional BoHV-1 vaccines, including those based on the use of modified live virus and also inactivated vaccines, are currently used in many countries, they have several disadvantages. DNA vaccines have emerged as an attractive approach since they have the potential to induce both humoral and cellular immune response; nevertheless, it is largely known that potency of naked DNA vaccines is limited. We demonstrated previously, in the murine model, that the use of adjuvants in combination with a DNA vaccine against BoHV-1 is immunologically beneficial. In this study, we evaluate the immune response and protection against challenge elicited in bovines, by a DNA vaccine carrying the sequence of secreted version of glycoprotein D (gD) of BoHV-1 formulated with chemical adjuvants. Bovines were vaccinated with formulations containing the sequence of gD alone or in combination with adjuvants ESSAI 903110 or Montanide™ 1113101PR. After prime vaccination and two boosters, animals were challenged with infectious BoHV-1. Formulations containing adjuvants Montanide™ 1113101PR and ESSAI 903110 were both, capable of increasing humoral immune response against the virus and diminishing clinical symptoms. Nevertheless, only formulations containing adjuvant Montanide™ 1113101PR was capable of improving cellular immune response and diminishing viral excretion. To our knowledge, it is the first time that a BoHV-1 DNA vaccine is combined with adjuvants and tested in cattle. These results could be useful to design a vaccine for the control of bovine rhinotracheitis.

Transient gene expression in serum-free suspension-growing mammalian cells for the production of foot-and-mouth disease virus empty capsids.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.

Immunogenicity of a bovine herpes virus I peptide expressed in tandem copies in attenuated Salmonella.

A live system to release heterologous antigens using an attenuated Salmonella strain was developed. We transformed Salmonella typhimurium LVR03 (S. LVR03) with a recombinant pTECH2 vector encoding 0, 1, 2, and 4 tandem copies of an imunogenic peptide of bovine herpes virus-1 (BoHV-1) glycoprotein D (gD). The system used yielded peptides fused to the non-toxic C fragment of the tetanus toxin (TetC), which has been shown to have adjuvant properties. Inoculation of BALB/c mice with the transformed Salmonella strains gave rise to a mild self-limited infection, with primary replication of bacteria occurring in Peyer's patches, even when the bacteria was administered intranasally. Humoral and cellular immune responses directed against the BoHV-1 antigens were evaluated after oral or intranasal administration of the recombinant bacteria. The results showed that the S. LVR03-dimer vaccine induced specific humoral (IgG in serum and IgG(1) and IgA in saliva), and cellular immune responses (lymphoproliferation and lymphokine secretion), against not only the selected peptide and whole gD, but also against BoHV-1, when administered intranasally. This is the first time Salmonella has been used as an expression vector to induce immunity against BoHV-1. This work demonstrates the feasibility of using this antigen-release system and encourages future experimentation with a bovine experimental model.

Glyphosate-based herbicides produce teratogenic effects on vertebrates by impairing retinoic acid signaling.

The broad spectrum herbicide glyphosate is widely used in agriculture worldwide. There has been ongoing controversy regarding the possible adverse effects of glyphosate on the environment and on human health. Reports of neural defects and craniofacial malformations from regions where glyphosate-based herbicides (GBH) are used led us to undertake an embryological approach to explore the effects of low doses of glyphosate in development. Xenopus laevis embryos were incubated with 1/5000 dilutions of a commercial GBH. The treated embryos were highly abnormal with marked alterations in cephalic and neural crest development and shortening of the anterior-posterior (A-P) axis. Alterations on neural crest markers were later correlated with deformities in the cranial cartilages at tadpole stages. Embryos injected with pure glyphosate showed very similar phenotypes. Moreover, GBH produced similar effects in chicken embryos, showing a gradual loss of rhombomere domains, reduction of the optic vesicles, and microcephaly. This suggests that glyphosate itself was responsible for the phenotypes observed, rather than a surfactant or other component of the commercial formulation. A reporter gene assay revealed that GBH treatment increased endogenous retinoic acid (RA) activity in Xenopus embryos and cotreatment with a RA antagonist rescued the teratogenic effects of the GBH. Therefore, we conclude that the phenotypes produced by GBH are mainly a consequence of the increase of endogenous retinoid activity. This is consistent with the decrease of Sonic hedgehog (Shh) signaling from the embryonic dorsal midline, with the inhibition of otx2 expression and with the disruption of cephalic neural crest development. The direct effect of glyphosate on early mechanisms of morphogenesis in vertebrate embryos opens concerns about the clinical findings from human offspring in populations exposed to GBH in agricultural fields.

Improved transfection of spleen-derived antigen-presenting cells in culture using TATp-liposomes.

Antigen presenting cells (APC) are among the most important cells of the immune system since they link the innate and the adaptative immune responses, directing the type of immune response to be elicited. To modulate the immune response in immune preventing or treating therapies, gene delivery into immunocompetent cells could be used. However, APC are very resistant to transfection. To increase the efficiency of APC transfection, we have used liposome-based lipoplexes additionally modified with cell-penetrating TAT peptide (TATp) for better intracellular delivery of a model plasmid encoding for the enhanced-green fluorescent protein (pEGFP). pEGFP-bearing lipoplexes made of a mixture of PC:Chol:DOTAP (60:30:10 molar ratio) with the addition of 2% mol of polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate (plain-L) or TATp-PEG-PE (TATp-L) were shown to effectively protect the incorporated DNA from degradation. Uptake assays of rhodamine-labeled lipoplexes and transfections with the EGFP reporter gene were performed with APC derived from the mouse spleen. TATp-L-based lipoplexes allowed for significantly enhanced both, the uptake and transfection in APC. Such a tool could be used for the APC transfection as a first step in immune therapy.