Synergism/complementarity of recombinant adenoviral vectors and other vaccination platforms during induction of protective immunity against malaria

The lack of immunogenicity of most malaria antigens and the complex immune responses required for achieving protective immunity against this infectious disease have traditionally hampered the development of an efficient human malaria vaccine. The current boom in development of recombinant viral vectors and their use in prime-boost protocols that result in enhanced immune outcomes have increased the number of malaria vaccine candidates that access pre-clinical and clinical trials. In the frontline, adenoviruses and poxviruses seem to be giving the best immunization results in experimental animals and their mutual combination, or their combination with recombinant proteins (formulated in adjuvants and given in sequence or being given as protein/virus admixtures), has been shown to reach unprecedented levels of anti-malaria immunity that predictably will be somehow reproduced in the human setting. However, all this optimism was previously seen in the malaria vaccine development field without many real applicable results to date. We describe here the current state-of-the-art in the field of recombinant adenovirus research for malaria vaccine development, in particular referring to their use in combination with other immunogens in heterologous prime-boost protocols, while trying to simultaneously show our contributions and point of view on this subject.

Efecte de la nutrició precoç en el desenvolupament de sibilàncies

Objectiu: provar que, enfront de l’aparició de sibilàncies, l’alletament matern es comporta com a un factor protector i l’alletament artificial com a un factor inductor. Material i mètodes: assaig clínic controlat, randomitzat, a doble cec amb grup control i seguiment de 8 anys, de la submostra espanyola, en el seu 5è any de seguiment, del treball multicèntric europeu EU CHILDHOOD OBESITY PROGRAMME (QLK1-2001-00389). La població es va dividir en 3 grups: nadons alimentats amb lactància artificial amb baix contingut proteic, nadons alimentats amb lactància artificial amb alt contingut proteic i un grup control de nadons alimentats amb llet materna. Per avaluar l’aparició de sibilàncies i la seva evolució en el temps es van realitzar entrevistes als pares a mesura que la població assolia els 6 anys de vida sobre qüestions referides als 3 i als 6 anys i s’havien de realitzar entrevistes als 8 anys de vida sobre qüestions referdies a aquesta mateixa edat. Per comprovar la repercussió en la funció pulmonar i valorar la base atòpica, es tenia previst realitzar, als 8 anys, espirometria, prik test amb aeroalergens, determinació de IgE sèrica total i quantificació dels eosinòfils en sang perifèrica. S’han valorat possibles factors de confusió com antecedents familiars de malalties de base al•lèrgica, nivell socioeconòmic familiar, factors, ambient epidemiològic i s’ha estudiat altra morbiditat associada com episodis de febre, vòmits, diarrea, dermatitis atòpica, refredat de vies respiratòries altes i prescripció mèdica d’antibiòtics. Resultats: només un 20’8% van rebre alletament matern. No s’han trobat diferències estadísticament significatives entre la història d’episodis de sibilàncies i el tipus d’alletament rebut. Tampoc s’han trobat diferències estadísticament significatives entre l’alimentació rebuda i la història de dermatitis atòpica. La llet artificial es va associar, amb significació estadística, a una major prescripció d’antibiòtics i una major incidència de patir diarrees i, sense significació estadística, es va associar a un augment del risc de patir RVA. La lactància materna es va associar amb significació estadística a una menor prescripció d’antibiòtics. La presència de germans grans i un baix nivell d’educació de la mare van contribuir a augmentar la morbiditat durant el primer any de vida. El consum d’alcohol durant l’embaràs es va associar a més episodis de vòmits i el consum de tabac a més episodis de diarrea. Conclusions: l’alletament artificial no predisposa a patir més episodis de sibilàncies ni de dermatitis atòpica. La lactància materna exclusiva durant almenys 3 mesos disminueix el risc de diarrees en els primers 6 mesos de vida i retarda l’aparició d’infeccions aparentment bacterianes que requereixen tractament antibiòtic. L’alletament matern exclusiu durant un mínim de tres mesos no comporta una substancial disminució de la morbiditat durant els primers 12 mesos de vida.

Production of TNF-α, nitric oxide and hydrogen peroxide by macrophages from mice with paracoccidioidomycosis that were fed a linseed oil-enriched diet

Omega-3 polyunsaturated fatty acids (n-3 PUFA) can modulate the immune system and their primary effect is on macrophage function. Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America that is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). Macrophages are the main defence against this pathogen and have microbicidal activity that is dependent on interferon-Γ and tumour necrosis factor (TNF)-α. These cytokines stimulate the synthesis of nitric oxide (NO) and hydrogen peroxide (H2O2), leading to the death of the fungus. To study the effect of n-3 PUFA on the host immune response during experimental PCM, macrophages that were obtained from animals infected with Pb18 and fed a diet enriched by linseed (LIN) oil were cultured and challenged with the fungus in vitro. The macrophage function was analysed based on the concentrations of TNF-α, NO and H2O2. LIN oil seems to influence the production of TNF-α during the development of disease. A diet enriched with LIN oil influences the microbicidal activity of the macrophages by inducing the production of cytokines and metabolites such as NO and H2O2, predominantly in the chronic phase of infection.

Efficient in vivo induction of CTL by cell-associated covalent H-2Kd-peptide complexes.

A novel procedure is presented describing the induction of antigen-specific cytolytic T lymphocytes (CTL) in vivo, that uses as immunogen syngeneic Concanavalin A stimulated spleen cells expressing H-2Kd (Kd) molecules photocrosslinked with a photoreactive peptide derivative. The Kd restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was conjugated with photoreactive iodo-4-azidosalicylic acid (IASA) at the NH2-terminus and with 4-azidobenzoic acid (ABA) at the TCR contact residue Lys259 to make IASA-YIPSAEK(ABA)I. Selective photoactivation of the IASA group allowed specific photoaffinity labeling of cell-associated Kd molecules. Optimal peptide derivative binding to Kd molecules of concanavalin A stimulated spleen cells was obtained upon 4-6 h incubation at 26 degrees C in the presence of human beta 2 microglobulin. Photocrosslinking prevented the rapid dissociation of cell-associated Kd-peptide derivative complexes at 37 degrees C. The photoaffinity labeled cells were injected i.p. into syngeneic recipients. After 10 days, the peritoneal exudate lymphocytes were harvested and in vitro stimulated with peptide derivative pulsed P815 mastocytoma cells. The resulting bulk cultures displayed high cytolytic activity that was specific for IASA-YIPSAEK(ABA)I and YIPSAEK(ABA)I. In contrast, peritoneal exudate lymphocytes from mice inoculated with concanavalin A blasts that were pulsed, but not photocrosslinked, with IASA-YIPSAEK(ABA)I expressed only marginal levels of IASA-YIPSAEK(ABA)I-specific cytolytic activity. This immunization strategy, using neither adjuvants nor potentially hazardous transfected/transformed cells, is safe and should be universally applicable.

In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression.

Via a transcription factor, Foxp3, immunoregulatory CD4(+)CD25(+) T cells (T reg cells) play an important role in suppressing the function of other T cells. Adoptively transferring high numbers of T reg cells can reduce the intensity of the immune response, thereby providing an attractive prospect for inducing tolerance. Extending our previous findings, we describe an in vivo approach for inducing rapid expansion of T reg cells by injecting mice with interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb). Injection of these IL-2-IL-2 mAb complexes for a short period of 3 d induces a marked (>10-fold) increase in T reg cell numbers in many organs, including the liver and gut as well as the spleen and lymph nodes, and a modest increase in the thymus. The expanded T reg cells survive for 1-2 wk and are highly activated and display superior suppressive function. Pretreating with the IL-2-IL-2 mAb complexes renders the mice resistant to induction of experimental autoimmune encephalomyelitis; combined with rapamycin, the complexes can also be used to treat ongoing disease. In addition, pretreating mice with the complexes induces tolerance to fully major histocompatibility complex-incompatible pancreatic islets in the absence of immunosuppression. Tolerance is robust and the majority of grafts are accepted indefinitely. The approach described for T reg cell expansion has clinical potential for treating autoimmune disease and promoting organ transplantation.

Two mechanisms of caspase 9 processing in double-stranded RNA- and virus-triggered apoptosis.

Viral double-stranded RNA (dsRNA) is a ubiquitous intracellular "alert signal" used by cells to detect viral infection and to mount anti-viral responses. DsRNA triggers a rapid (complete within 2-4 h) apoptosis in the highly-susceptible HeLa cell line. Here, we demonstrate that the apical event in this apoptotic cascade is the activation of procaspase 8. Downstream of caspase 8, the apoptotic signaling cascade bifurcates into a mitochondria-independent caspase 8/caspase 3 arm and a mitochondria-dependent, caspase 8/Bid/Bax/Bak/cytochrome c arm. Both arms impinge upon, and activate, procaspase 9 via two different cleavage sites within the procaspase 9 molecule (D330 and D315, respectively). This is the first in vivo demonstration that the "effector" caspase 3 plays an "initiator" role in the regulation of caspase 9. The dsRNA-induced apoptosis is potentiated by the inhibition of protein synthesis, whose role is to accelerate the execution of all apoptosis steps downstream of, and including, the activation of caspase 8. Thus, efficient apoptosis in response to viral dsRNA results from the co-operation of the two major apical caspases (8 and 9) and the dsRNA-activated protein kinase R (PKR)/ribonuclease L (RNase L) system that is essential for the inhibition of protein synthesis in response to viral infection.

Prevention of atelectasis formation during the induction of general anesthesia in morbidly obese patients

Résumé: La formation des atélectasies durant l'induction de l'anesthésie générale est plus importante chez le patient obèse morbide. Nous avons démontré dans des travaux de recherche antérieurs que l'utilisation de la PEEP (Pression Positive en Fin d'Expiration) durant l'induction de l'anesthésie prévient la formation d'atélectasies chez des patients non obèses. Par conséquent, nous voulions étudier l'efficacité de la pression positive en fin d'expiration chez le patient obèse morbide dans la prévention de la formation d'atélectasies. Nous avons fait une étude de 23 patients obèses morbides (BMI > 35 kg / m2) dans 2 groupes. Dans le groupe utilisant la pression positive en fin d'expiration, les patients respiraient 100% d'oxygène pendant 5 minutes par l'intermédiaire d'un masque facial type CPAP avec une pression de 10 cm H20. Après l'induction de l'anesthésie, nous avons ventilé les patients au masque facial avec une PEEP de 10 cm H20. Dans le groupe de contrôle, nous avons procédé au même type d'induction sans utiliser la pression positive en fin d'expiration. La surface de poumon atélectatique a été évaluée par tomographie (CT scann). L'étude des échanges gazeux se faisait à 2 reprises, à partir de gazométries réalisées juste avant l'induction de l'anesthésie puis juste après l'intubation. Après l'induction de l'anesthésie et l'intubation, les patients du groupe de contrôle présentaient une quantité d'atélectasies plus importante que les patients du groupe où la PEEP avait été utilisée (10.4% + 4.8% dans le groupe de contrôle versus 1.3% dans le groupe utilisant la pression positive en fin d'expiration p < 0.001). Après l'intubation, en présence d'une fraction inspirée en oxygène à 100%, la Pa02 était significativement supérieure dans le groupe ayant utilisé la pression positive en fin d'expiration en comparaison avec le groupe de contrôle (respectivement 457 ± 130 mmHg versus 315 ± 100 mmHg). Nous avons conclu que chez le patient obèse morbide, le recours à la pression positive en fin d'expiration lors de l'induction de l'anesthésie permet de prévenir largement la formation d'atélectasies et s'accompagne d'une meilleure oxygénation. Abstract: Atelectasis caused by general anesthesia is increased in morbidly obese patients. We have shown that application of positive end-expiratory pressure (PEEP) during the induction of anesthesia prevents atelectasis formation in nonobese patients. We therefore studied the efficacy of PEEP in morbidly obese patients to prevent atelectasis. Twenty-three adult morbidly obese patients (b ody mass index >35 kg/m2) were randomly assigned to one of two groups. In the PEEP group, patients breathed 100% oxygen (5 min) with a continuous positive airway pressure of 10 cm H20 and, after the induction, mechanical ventilation via a face mask with a PEEP of 10 cm H2O. In the control group, the same induction was applied but without continuous positive airway pressure or PEEP. Atelectasis, determined by computed tomography, and blood gas analysis were measured twice: before the induction and directly after intubation. After endotracheal intubation, patients of the control group showed an increase in the amount of atelectasis, which was much larger than in the PEEP group (10.4% -± 4.8% in control group versus 1.7% ± 1.3% in PEEP group; P <0.001). After in.tubation with a fraction of inspired oxygen of 1.0, Pao, was significantly higher in the PEEP group compared with the control group (457 ±- 130 mm Hg versus 315 ± 100 mm Hg, respectively; P = 0.035) We conclude that in morbidly obese patients, atelectasis formation is largely prevented by PEEP applied during the anesthetic induction and is associated with a better oxygenation.

The roles of PKCδ and Src kinases in the glucocorticoid induction and the TGF-β superinduction of the mouse mammary tumor virus transcription in B lymphocytes

Résumé : Le virus tumoral de la glande mammaire de la souris (MMTV) est un rétrovirus provoquant le développement de tumeurs dans les glandes mammaires des souris susceptibles femelles. Au cours de son évolution, le virus s'est adapté et s'exprime dans des cellules spécialisées. Les lymphocytes B sont les premières cellules infectées et elles sont essentielles pour la propagation de l'infection aux glandes mammaires. Dans notre étude, le virus MMTV a été utilisé afin d'examiner les voies de signalisation induites par les glucocorticoïdes (dexaméthasone (dex), une hormone stéroïdienne) et le transforming growth factor-f3 (TGF-P, une cytokine), deux molécules impliquées dans l'activation de la transcription à partir du promoteur du MMTV dans les cellules B. Le TGF-P seul n'influence pas l'activité du promoteur du MMTV. Par contre, en synergie avec dex, le TGF-P provoque une super-induction de l'expression du promoteur par rapport à une stimulation par le glucocorticoïde seul. Cette super-induction est régulée par une famille de protéines, les Smads. Ainsi, dans les lymphocytes B, l'utilisation du MMTV a permis de mettre en évidence une nouvelle synergie entre les glueocortieoïdes et le TGF-p. pans ce travail, l'utilisation d'inhibiteurs pharmacologiques et de mutants « dominant-négatifs » nous a pet mis de démontrer qu'une Protéine Kinase C delta (PKC5) active est impliquée dans la transduction du signal lors de la réponse au dex ainsi que celle au TGF-P. Néanmoins, la PKC5 est régulée différemment dans chaque voie spécifique : la voie du TGF-p nécessitait l'activation du PKC5 par diacylglycerol (DAG) et la phosphorylation de tyrosines spécifiques, alors que la voie impliquant les glucocorticoïdes ne le nécessitait pas. Nous avons aussi démontré qu'une tyrosine kinase de la famille Src est responsable de la phosphorylation des tyrosines sur la PKC5. Les essais de kinase in vitro nous ont permis de découvrir que plusieurs Src kinases peuvent phosphoryler la PKC6 dans les cellules B et qu'elles étaient constitutivement actives. Enfin, nous avons montré qu'il existe une interaction protéine - protéine induite par dex, entre le récepteur aux glucocorticoïdes (GR) et la PKC5 dans les cellules B, une association qui n'a pas été démontrée auparavant. Par ailleurs, nous avons analysé les domaines d'interactions entre PKC5 et GR en utilisant les essais de «GST pull-down». Nos résultats montrent que le domaine régulateur de la PKC5 et celui qui interagit avec l'ADN du GR sont impliqués. En résumé, nous avons trouvé que dans une lignée lymphocytaire B, le virus MMTV utilise des mécanismes pour réguler à la fois la transcription et la voie de signalisation qui sont différents de ceux utilisés dans les cellules mammaires épithéliales et les fibroblastes. Nos découvertes pourraient être utilisées comme modèles pour l'étude de gènes cellulaires impliqués dans des processus tels qu'inflammation, immunité ou cancérogénèse. Summary: Mouse Mammary Tumor Virus (MMTV) is a retrovirus that causes tumors in the mammary glands of susceptible female mice and has adapted evolutionarily to be expressed in specialized cells. The B lymphocytes are the first cells to be infected by the MMTV and are essential for the spread of infection to the mammary glands. Here, we used the MMTV as a model system to investigate the signalling cascade induced by giucocorticoids (dexamethasone, "dex", a steroid hormone), and by Transforming Growth Factor-beta (TGF-P, a cytokine) leading to its transcriptional activation in B lymphocytes. By itself, TGF-I3 does not affect the basal activity of the MMTV promoter. However, TGF-13 significantly increases glucocorticoid-induced expression, through its effectors, the Smad factors. Thus, MMTV in B cells demonstrates a novel synergism between glucocorticoids and TGF-16. In this thesis project, we present evidence, based on the use of pharmacological inhibitors and of dominant-negative mutants, that an active Protein Kinase C delta (PKC6) is required as a signal transducer for the dex response and for the TGF-P superinduction as well. The PKC6 is differentially regulated in each specific pathway: whereas the TGF-13 superinduction required PKC6 to be activated by diacylglycerol (DAG) and to be phosphorylated at specific tyrosine residues, the glueocorticoid-induced pathway did not. We also showed that a protein tyrosine kinase of the Src family is responsible for the phosphorylation of tyrosines on PKC6. By performing in vitro kinase assays, we found that several Src kinases of B cells were able to phosphorylate PKC6 and that they were constitutively active. Finally, we demonstrate a dex-dependent functional protein-protein interaction between the glucocorticoid receptor (GR) and PKC6 in B cells, an association that has not been previously described. We further analysed the interacting domains of PKG6 and GR using in vitro GST pull-down assays, whereby the regulatory domain of PKC6 and the extended DNA-binding domain of the GR were involved. In summary, we found that in B-lymphoid cell lines, MMTV uses novel mechanisms of transcriptional control and signal transduction that are different from those at work in mammary epithelial or fibroblastic cells. These findings will be used as model for cellular genes involved in cellular processes such as immune functions, inflammation, or oncogenic transformation that may have a similar pattern of regulation.

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

An empirical study of machine learning techniques applied to image watermarking

A study of how the machine learning technique, known as gentleboost, could improve different digital watermarking methods such as LSB, DWT, DCT2 and Histogram shifting.