A recombinant influenza virus vaccine expressing the F protein of respiratory syncytial virus.

Infections with influenza and respiratory syncytial virus (RSV) rank high among the most common human respiratory diseases worldwide. Previously, we developed a replication-incompetent influenza virus by replacing the coding sequence of the PB2 gene, which encodes one of the viral RNA polymerase subunits, with that of a reporter gene. Here, we generated a PB2-knockout recombinant influenza virus expressing the F protein of RSV (PB2-RSVF virus) and tested its potential as a bivalent vaccine. In mice intranasally immunized with the PB2-RSVF virus, we detected high levels of antibodies against influenza virus, but not RSV. PB2-RSVF virus-immunized mice were protected from a lethal challenge with influenza virus but experienced severe body weight loss when challenged with RSV, indicating that PB2-RSVF vaccination enhanced RSV-associated disease. These results highlight one of the difficulties of developing an effective bivalent vaccine against influenza virus and RSV infections.

Structural and functional analysis of the Entamoeba histolytica EhrabB gene promoter.

BACKGROUND: The Entamoeba histolytica EhrabB gene encodes for a Rab GTPase involved in phagocytosis. It is located at a virulence locus where the Ehcp112 gene is in the complementary strand at 332 bp of EhrabB start codon, suggesting a finely regulated transcription of both genes. However, the transcription regulation in this parasite is poorly understood. RESULTS: To initiate the knowledge of EhrabB gene expression regulation, here we studied the structural characteristics of its gene promoter and its control transcription elements. In silico searches of the EhrabB 5'-flanking region revealed that it contains a motif similar to the upstream regulatory element 1 (URE1) of the E. histolytica hgl5 gene. It also has sequences with homology to C/EBP and GATA1 binding sites, and heat shock elements (HSE). Primer extension experiments revealed that EhrabB has at least four transcription initiation sites. The elements at the 5'-flanking region that drive EhrabB gene expression were detected and characterized using transitory transfected trophozoites with a plasmid carrying the CAT reporter gene. EhrabB transcription is negatively regulated by a sequence located between positions -491 to -428 with respect to the first transcription initiation site. We also showed that the URE1-like motif activates EhrabB transcription. In addition, heat shock activated the EhrabB promoter in episomal constructs and lead to an increase in de novo EhrabB transcription. CONCLUSION: The data suggest that EhrabB transcription is controlled negatively by an unidentified sequence, but it is activated by an URE1-like motif. Our analyses also revealed the presence of activator HSE that function under stress.

The effect of the administration of human gamma globulins in a model of BCG infection in mice.

The effect of the administration of a commercial preparation of human gamma globulins has been evaluated in a mouse model of intranasal infection with BCG. First, we demonstrated the passage of specific antibodies to saliva and lung lavage following the intranasal or intraperitoneal administration to mice of human gamma globulins. This treatment of mice inhibited BCG colonization of the lungs (p < 0.01). A similar inhibitory effect was observed after infection of mice with gamma globulin opsonized BCG organisms (p < 0.01). These results are relevant for the development of new strategies for the control and treatment of tuberculosis.

Relationship between presence of abfractions and premature contacts

ABSTRACT: Abfractions (AFs) are non-carious cervical lesions considered of multifactorial etiology by several authors. Objective: Relate the presence of premature contacts and AFs. Materials & Methods: The original number of students to be analyzed were 117, 36 of whom (equal amount of women and men) were finally chosen for presenting premature contacts. 19 of them presented abfractions. A survey was conducted with questions on issues like toothbrushing habits, diet and some gastric disorders; besides the participants had to take the Hamilton Anxiety Rating Scale. Then, stone models were obtained and mounted for further occlusal analysis with WCM ® semi-adjustable articulator searching the abfractions and their coincidence with premature contacts. Fisher's exact test (p<0,05) was used to associate AF with toothbrushing habits, diet, burning symptoms and gastroesophageal reflux. Chi-square test (p<0,05) was used to associate AFs and premature contacts. Student's t-distribution was used to associate AFs with anxiety. Results: There was a significant relationship between AFs presence and premature contacts (p=0,00). There was not a significant relationship between presence of AFs and toothbrushing habits and diet. There was not a significant relationship between presence of AFs and anxiety. Conclusions: AF presence do associate with premature contacts in the same affected tooth.

Effect of the silencing of the Ehcp112 gene on the in vitro virulence of Entamoeba histolytica.

BACKGROUND: Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis in humans, affecting up to 50 million people worldwide each year and causing 40,000 to 100,000 deaths annually. EhCP112 is a cysteine proteinase of E. histolytica able to disrupt cell monolayers and digest extracellular matrix proteins, it is secreted by trophozoites and it can be active in a wide range of temperature and pH. These characteristics have encouraged the use of EhCP112 in the design and production of possible vaccines against amoebiasis, obtaining promising results. Nevertheless, we have no conclusive information about the role of EhCP112 in the E. histolytica pathogenesis. METHODS: A set of three specific siRNA sequences were used to silence the Ehcp112 gene via the soaking system. Silencing was evaluated by Western blot using an antibody against the EhCP112 recombinant protein. Finally, we analyzed the protease activity, the phagocytosis rate and the ability to destroy MDCK cells of the EhCP112-silenced trophozoites. RESULTS: The highest silencing effect on EhCP112 was detected at 16 h of treatment; time enough to perform the in vitro virulence assays, which showed that EhCP112 silencing produces a significant reduction in cytolysis and phagocytosis of target cells, indicating the participation of this proteinase in these events. CONCLUSIONS: EhCP112 is involved in the in vitro virulence of E. histolytica.

A high-throughput drug screen for Entamoeba histolytica identifies a new lead and target.

Entamoeba histolytica, a protozoan intestinal parasite, is the causative agent of human amebiasis. Amebiasis is the fourth leading cause of death and the third leading cause of morbidity due to protozoan infections worldwide(1), resulting in ~70,000 deaths annually. E. histolytica has been listed by the National Institutes of Health as a category B priority biodefense pathogen in the United States. Treatment relies on metronidazole(2), which has adverse effects(3), and potential resistance of E. histolytica to the drug is an increasing concern(4,5). To facilitate drug screening for this anaerobic protozoan, we developed and validated an automated, high-throughput screen (HTS). This screen identified auranofin, a US Food and Drug Administration (FDA)-approved drug used therapeutically for rheumatoid arthritis, as active against E. histolytica in culture. Auranofin was ten times more potent against E. histolytica than metronidazole. Transcriptional profiling and thioredoxin reductase assays suggested that auranofin targets the E. histolytica thioredoxin reductase, preventing the reduction of thioredoxin and enhancing sensitivity of trophozoites to reactive oxygen-mediated killing. In a mouse model of amebic colitis and a hamster model of amebic liver abscess, oral auranofin markedly decreased the number of parasites, the detrimental host inflammatory response and hepatic damage. This new use of auranofin represents a promising therapy for amebiasis, and the drug has been granted orphan-drug status from the FDA.

A pcDNA-Ehcpadh vaccine against Entamoeba histolytica elicits a protective Th1-like response in hamster liver.

DNA vaccines are promising tools to fight parasitic diseases, including amoebiasis caused by the protozoan Entamoeba histolytica. Here we studied the immunogenicity and protective efficacy of a DNA vaccine against this parasite composed by the EhCPADH surface complex encoding genes (Ehcp112 and Ehadh112). EhCPADH is formed by an adhesin (EhADH112) and a cysteine proteinase (EhCP112), both involved in the parasite virulence. We evaluated transcription, protein expression, immunological response and protection against hepatic amoebiasis in hamsters intradermally and intramuscularly immunized with a mixture of pcDNA-Ehadh112 and pcDNA-Ehcp112 plasmids. RT-PCR and immunohistochemical assays showed that both antigens were differentially expressed in spleen and liver of immunized animals. No significant antibody immune response was induced by either route. However, intradermally inoculated hamsters presented a robust Th1-like immune response, characterized by high levels of INF-gamma and TNF-alpha cytokines, detected in the liver of animals challenged with virulent trophozoites. Animals showed significant protection against amoebiasis manifested by a higher survival rate and a significant prevention of liver abscess formation. We conclude that a refinement of this DNA vaccine could be a good choice to control hepatic amoebiasis.

Expression in fibroblasts and in live animals of Entamoeba histolytica polypeptides EhCP112 and EhADH112.

EhCPADH is an immunogenic, heterodimeric protein that is formed by EhCP112 (cysteine protease) and EhADH112 (adhesin), polypeptides involved in Entamoeba histolytica's cytopathic effect, target-cell adherence and phagocytosis. The EhCPADH complex is located in the plasma membrane and cytoplasmic vacuoles. Here, the independent expression of EhCP112 and EhADH112 in fibroblasts and hamsters was analysed. Also investigated was the immunological response in animals independently inoculated with plasmid pcDNA-Ehcp112, which carries the complete cysteine protease-encoding gene, or with plasmid pcDNA-Ehadh112, which carries the C terminus of the adhesin-encoding gene, or with a mixture of both. Both proteins were expressed in the plasma membranes of the transfected fibroblasts. EhCP112 was toxic for the mammalian cells. Proteins were also independently expressed in hamsters after inoculation with the plasmids. Their expression was indirectly evaluated by the presence of antibodies in the inoculated animals. Remarkably, co-immunization of the animals with the two DNA plasmids resulted in an earlier and higher anti-E. histolytica IgG induction than immunization with separate plasmids. In contrast, the cellular immune response was not noticeably improved by the plasmid mixture. Interestingly, protection against liver abscesses was detected only in animals that received the plasmid mixture and no protection was observed in hamsters independently inoculated with plasmid pcDNA-Ehcp112 or pcDNA-Ehadh112.

Desarrollo de biomodelos para la evaluación de la inmunidad secretora contra M. tuberculosis en ratones Babl/c / Development of Biomodels for the evaluation of the secretory immunity against M tuberculosis in Balb/c mice

Poco se ha estudiado acerca del papel de los anticuerpos específicos, presentes en las secreciones del aparato respiratorio, en la defensa contra patógenos intracelulares, como es el caso de las micobacterias causantes de la tuberculosis en el hombre: Micobacterium tuberculosis, bovis y africanum. Con el objetivo de desarrollar modelos adecuados para evaluar el posible papel de la inmunidad secretoria en la defensa contra la tuberculosis, se desarrollaron dos modelos animales con la utilización de un anticuerpo monoclonal IgA dirigido contra la proteína de 16 kD de M. tuberculosis y M. bovis. En el primer modelo se inocularon ratones Balb/c, por vía subcutánea al nivel de la nuca, con diferentes cantidades de células del hibridoma TBA61, productor de la IgA específica. En un segundo modelo, se inoculó por vía intraperitoneal líquido ascítico correspondiente a este hibridoma obtenido en ratón. En ambos casos se determinó, a diferentes tiempos, la concentración del monoclonal en saliva y sólo en suero para el segundo. En los dos modelos se demostró el paso del monoclonal a la saliva, donde alcanzó la máxima concentración: a los 21 días en los animales inoculados con el hibridoma, y a las 2 horas en saliva y suero en los animales inoculados con el líquido ascítico. Se sugiere, por su sencillez y mayor inocuidad, el uso del segundo modelo para la realización de estudios de reto por vía mucosal(AU)

Antibióticos nebulizados en pacientes con fibrosis quística / Nebulized antibiotics in patients with cystic fibrosis

No disponible