Dengue vaccine: an update on recombinant subunit strategies.

Dengue is an increasing public health problem worldwide, with the four serotypes of the virus infecting over 390 million people annually. There is no specific treatment or antiviral drug for dengue, and prevention is largely limited to controlling the mosquito vectors or disrupting the human-vector contact. Despite the considerable progress made in recent years, an effective vaccine against the virus is not yet available. The development of a dengue vaccine has been hampered by many unique challenges, including the need to ensure the absence of vaccine-induced enhanced severity of disease. Recombinant protein subunit vaccines offer a safer alternative to other vaccine approaches. Several subunit vaccine candidates are presently under development, based on different structural and non-structural proteins of the virus. Novel adjuvants or immunopotentiating strategies are also being tested to improve their immunogenicity. This review summarizes the current status and development trends of subunit dengue vaccines.

A CFSE-based assay of proliferative response of lymphocytes to stimulation with dengue viruses.

Dengue viruses (DENVs) are human pathogens that constitute a significant threat worldwide. Since they up-regulate MHC class I molecules; the cell-mediated immunity may play an important role in the defense against viruses. In this work, we tested a CFSE-based assay in determining proliferative response of lymphocytes isolated from mice or monkeys previously immunized with various DENV antigens to in vitro stimulation with DENVs. A positive proliferative response was obtained with lymphocytes of animals immunized with either live DENV-2 or its recombinant proteins. A similar result was also obtained with CD8+ T cells from mice immunized with live DENV-1 or DENV-2 following stimulation with homologous viruses. A comparison of the carboxyfluorescein diacetate succinimidyl ester (CFSE)-based and a 3H-thymidine incorporation-based assays of proliferative response of total lymphocytes showed a fair correlation of results of both assays. However, the CSFE-based assay offers in addition the determination of contribution of the CD8+ or other subsets of T cells to total proliferative response. These results represent the first and successful application of a CFSE-based assay to the evaluation of cell-mediated immunity to DENVs. This assay might be also exploited in testing candidate DENV vaccines.

GermOnline, a cross-species community knowledgebase on germ cell differentiation.

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.

Inhaled nitric oxide through a noninvasive ventilation device to assess reversibility of pulmonary hypertension in selecting recipients for heart transplant.

INTRODUCTION: Pulmonary hypertension (PHT) is an independent risk factor for right ventricular failure and death after heart transplant. Nitric oxide (NO) is a powerful and selective vasodilator, indicated in this scenario, but its response is unpredictable. Thus, it should be assessed prior to the intervention. However, preoperative assessment has not been widespread due to its difficulties and risks. OBJECTIVE: We describe herein a pulmonary vasodilatory test with NO administered through a noninvasive ventilation (NIMV) device. We also assessed the effect of NO in patients with severe PHT owing to cardiac disease. Assessment of the utility of the test to select patients for heart transplant. METHODS: We enrolled 19 patients with severe PHT for a preoperative assessment for heart transplant. Thresholds used were as follows: systolic pulmonary arterial pressure (SPAP) > or =65 mm Hg, transpulmonary gradient (TPG) > or =15 mm Hg, and pulmonary vascular resistance (PVR) > or =4.5 Wood units (WU). NO was administered through a modified noninvasive ventilation device. Cardiac output and pulmonary pressures were measured simultaneously by right heart catheterization. RESULTS: All patients agreed to be enrolled in the test. No difficulties, interruptions, or severe complications happened in any case. Basal and NO average measured values were SPAP (74.16 and 57.95 mm Hg), PVR (7.5 and 3.7 WU), and TPG (23.25 and 12.58 mm Hg). The differences were significant (P < .05) for all three tests. We consider acceptable for heart transplant a response that reduces PHT to a moderate grade. Using these criteria 14 patients were accepted and 11 underwent heart transplant. Two deaths in the postoperative period were both secondary to mediastinal bleeding and not related to right ventricular failure. CONCLUSIONS: A pulmonary vasodilatory test with NO administered through a NIMV device was feasible and useful to select suitable heart transplant recipients with severe pulmonary hypertension.

Long-term results of heart transplant in recipients older and younger than 65 years: a comparative study of mortality, rejections, and neoplasia in a cohort of 445 patients.

BACKGROUND: Whether being older than 65 years should be considered an absolute counterindication to heart transplant (HT), as it is in some centers, is controversial. In our centre, patients older than 65 years are accepted for HT if they satisfy stringent conditions. The aim of this study was to examine whether heart recipients older than 65 years have a greater risk of rejection, neoplasia, or mortality than younger ones. METHODS: We studied 445 patients who underwent HT between April 1991 and December 2003, 42 of whom were older than 65 years and 403 who were 65 years or younger. The parameters evaluated were the cumulative incidences of neoplasias and rejections (ISHLT grade > or = 3A), and the survival rates 1 month, 1 year, and 5 years post-HT. RESULTS: The two groups had similar percentages of patients with at least one rejection episode (< or =65 years 56.9%, >65 years 51.3%; P > .05), and although there were proportionally almost twice as many tumors in the older group (14.2%) as in the younger (7.9%), this difference was not statistically significant either. Nor were there any significant differences in survival, the 1-month, 1-year, and 5-year rates being 87.8%, 82.1%, and 68.8%, respectively, in the younger group and 85.7%, 78.6%, and 73.4%, respectively, in the older. CONCLUSIONS: Among carefully selected patients aged more than 65 years, HT can be performed without incurring greater risk of rejection, malignancy, or death than is found among recipients younger than 65 years.

Absence of enteroviral RNA in hearts explanted from patients with dilated cardiomyopathy.

BACKGROUND: The role of enterovirus infection in the pathogenesis of dilated cardiomyopathy (DCM) remains unclear. The objective of this study was to determine the prevalence of enterovirus in hearts explanted from patients with DCM and to compare it with enterovirus prevalence in hearts explanted from patients with other etiologies and in healthy donor hearts. METHODS: A total of 138 cardiac samples were analyzed, 70 from heart donors and 68 from transplant recipients (22 with DCM). A highly sensitive enterovirus-specific nested RT-PCR was used to test for enterovirus. RESULTS: All tests were negative except for one positive result that was attributed to carryover because sequencing of the amplification product showed it to be identical to the positive control. CONCLUSIONS: In this study the sample of explanted hearts nested RT-PCR showed no evidence of the presence of enteroviral RNA. This suggests that if enterovirus had a role in the genesis of DCM, it does not require or lead to the persistence of the virus in myocardial tissue.

A recombinant envelope protein from Dengue virus purified by IMAC is bioequivalent with its immune-affinity chromatography purified counterpart.

Semi-purified DEN-4 envelope protein, obtained in Pichia pastoris, was capable of generating neutralising and protecting antibodies after immunisation in mice. Here we compared two purification processes of this recombinant protein using two chromatographic steps: immune-affinity chromatography and immobilised metal ion adsorption chromatography (IMAC). The protein purified by both methods produced functional antibodies reflected by titres of haemagglutination inhibition and neutralisation. IMAC could be used as an alternative for high scale purification.

Analysis of cis-trans isomers and enantiomers of sertraline by cyclodextrin-modified micellar electrokinetic chromatography.

In this work development, optimization and validation of a cyclodextrin-modified micellar electrokinetic chromatography (CD-modified MEKC) method is proposed to resolve separation of the sertraline hydrochloride and synthesis-related substances. Sertraline hydrochloride, the cis-(1S,4S) enantiomer form, is used as an antidepressant therapeutic agent. A buffer concentration composed of 20 mM sodium borate, pH 9.0 with 50 mM sodium cholate, 15 mM sulfated beta-cyclodextrin and 5 mM hydroxypropyl-beta-cyclodextrin was found to be the most suitable background electrolyte. Quantitation of the impurities at levels of 0.1% in different samples of the bulk drug was determined. A comparison of the results with those obtained by HPLC methodology was also accomplished. The method proved appropriate for testing the purity of sertraline hydrochloride in bulk drug.

Chylous ascites following heart transplantation.

We report a case of chylous ascites in a 52-year-old woman after an orthotopic heart transplantation. The patient was successfully managed with conservative treatment including dietetic measures and repeated paracentesis. The potential aetiopathological factors involved and other implications for cardiac transplant patients are discussed.

Evaluation of anti-Moraxella bovis pili immunoglobulin-A in tears following intranasal vaccination of cattle.

Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease of cattle caused by Moraxella bovis (Mb). Parenterally administered immunogens used to prevent the disease do not offer complete protection possibly because they stimulate a poor ocular mucosal secretory response, in which locally secreted immunoglobulin-A (sIgA) is one of the main components. The principal aim of this study was to evaluate by an indirect enzyme linked immunosorbent assay (ELISA), the local ocular mucosal sIgA response against Mb purified pili, produced after intranasal inoculation of experimental vaccines. Pili were adjuvanted by several different adjuvants (QuilA, Marcol Arlacel, Marcol Span, microencapsulated pili with PLGA polymers). Results were compared to sIgA response produced by adjuvant placebo inoculations and by IBK natural infection. Significantly higher anti-pili IgA response (p<0.05) was detected in calves vaccinated intranasally with pili QuilA and pili Marcol Span compared to control calves, although this specific immune response did not seem to be related to protection against Mb infection or typical IBK lesion development.

Microencapsulated citronella oil for mosquito repellent finishing of cotton textiles.

Microcapsules containing citronella essential oil were prepared by complex coacervation and applied to cotton textiles in order to study the repellent efficacy of the obtained fabrics. Citronella released from treated textiles was indirectly monitored by the extractable content of its main components. Repellent activity was assessed by exposure of a human hand and arm covered with the treated textiles to Aedes aegypti mosquitoes. Fabrics treated with microencapsulated citronella presented a higher and longer lasting protection from insects compared to fabrics sprayed with an ethanol solution of the essential oil, assuring a repellent effect higher than 90% for three weeks. Complex coacervation is a simple, low cost, scalable and reproducible method of obtaining encapsulated essential oils for textile application. Repellent textiles were achieved by padding cotton fabrics with microcapsules slurries using a conventional pad-dry method. This methodology requires no additional investment for textile finishing industries, which is a desirable factor in developing countries.