Immunological and protective properties of novel malaria blood stage peptide antigens

ABSTRACT Malaria is a major worldwide public health problem, with transmission occurring throughout Africa, Asia, Oceania and Latin America. Over two billion people live in malarious areas of the world and it is estimated that 300-500 million cases and 1.5-2.7 million deaths occur annually. The increase in multi-drug resistant parasites and insecticide-resistant vectors has made the development of malaria vaccine a public health priority. The published genome offers tremendous opportunity for the identification of new antigens that can befast-tracked for vaccine development. We identified potential protein antigens present on the surface of asexual malaria blood stages through bioinformatics and published transcriptome and proteorné analysis. Amongst the proteins identified, we selected those that contain predicted a-helical coiled-coil regions, which are generally short and structurally stable as isolated fragments. Peptides were synthesized and used to immunize mice. Most peptides tested were immunogenic as demonstrated in ELISA assays, and induced antibodies of varying titres. In immunofluorescence assays, anti-sera from immunized mice reacted with native proteins expressed at different intraerythrocytic developmental stages of the parasite's cycle. In parallel in vitro ADCI functional studies, human antibodies affinity purified on some of these peptides inhibited parasite growth in association with monocytes in magnitudes similar to that seen in semiimmune African adults. Siudies using human immune sera taken from different malaria endemic regions, demonstrated that majority of peptides were recognized at high prevalence. 73 peptides were next tested in longitudinal studies in two cohorts separated in space and time in coastal Kenya. In these longitudinal analyses, antibody responses to peptides were sequentially examined in two cohorts of children at risk of clinical malaria in order to characterize the level of peptide recognition by age, and the role of anti-peptide antibodies in protection from clinical malaria. Ten peptides were associated ?with a significantly reduced odds ratio for an episode of clinical malaria in the first cohort of children and two of these peptides (LR146 and ÁS202.11) were associated with a significantly reduced odds ratio in both cohorts. This study has identified proteins PFB0145c and MAL6P1.37 among others as likely targets of protective antibodies. Our findings support further studies to systematically assess immunogenicity of peptides of interest in order to establish clear criteria for optimal design of potential vaccine constructs to be tested in clinical trials. RESUME La malaria est un problème de santé publique mondial principalement en Afrique, en Asie, en Océanie et en Amérique latine. Plus de 2 milliards de personnes vivent dans des régions endémiques et le nombre de cas par année est estimé entre 300 et 500 millions. 1.5 à 2.7 millions de décès surviennent annuellement dans ces zones. L'augmentation de la résistance aux médicaments et aux insecticides fait du développement d'un vaccin une priorité. Le séquençage complet du génome du parasite offre l'opportunité d'identifier de nouveaux antigènes qui peuvent rapidement mener au développement d'un vaccin. Des protéines antigéniques potentielles présentes à la surface des globules rouges infectés ont été identifiées par bioinformatique et par l'analyse du protéome et du transcriptome. Nous avons sélectionné, parmi ces protéines, celles contenant des motifs dits "a helical coiled-coil" qui sont généralement courts et structurellement stables. Ces régions ont été obtenues par synthèse peptidique et utilisées pour immuniser des souris. La plupart des peptides testés sont immunogéniques et induisent un titre variable d'anticorps déterminé par ELISA. Les résultats de tests d'immunofluorescence indiquent que les sera produits chez la souris reconnaissent les protéines natives exprimées aux différents stades de développement du parasite. En parallèle, des études d'ADCI in vitro montrent qué des anticorps humains purifiés à partir de ces peptides associés à des monocytes inhibent la croissance du parasite aussi bien que celle observée chez des adultes africains protégés. Des études d'antigénicité utilisant des sera de personnes protégées de différents âges vivant dans des régions endémiques montrent que la majorité des peptides sont reconnus avec une haute prévalence. 73 peptides ont été testés dans une étude longitudinale avec 2 cohortes de la côte du Kenya. Ces 2 groupes viennent de zones bien distinctes et les prélèvements n'ont pas été effectués pendant la même période. Dans cette étude, la réponse anticorps contre les peptides synthétiques a été testée dans les 2 cohortes d'enfants à risque de développer un épisode de malaria afin de caractériser le niveau de reconnaissance des peptides en fonction de l'âge et de déterminer le rôle des anticorps anti-peptides dans la protection contre la malaria. Parmi ces peptides, 10 sont associés à une réduction significative des risques de développer un épisode de malaria dans la première cohorte alors qu'un seul (LR146 et AS202.11) l'est dans les 2 cohortes. Cette étude a identifié, parmi d'autres, les protéines PFB0145c et MAL6P1.37 comme pouvant être la cible d'anticorps. Ces résultats sont en faveur de futures études qui évalueraient systématiquement l'immunogénicité des peptides d'intérêt dans le but d'établir des critères de sélection clairs pour le développement d'un vaccin. Résumé pour un large public La malaria est un problème de santé publique mondial principalement en Afrique, en Asie, en Océanie et en Amérique latine. Plus de 2 milliards de personnes vivent dans des régions endémiques et le nombre de cas par année est estimé entre 300 et 500 millions. 1.5 à 2.7 millions de décès surviennent annuellement dans ces zones. La résistance aux médicaments et aux insecticides augmente de plus en plus d'où la nécessité de développer un vaccin. Le séquençage complet du génome (ensemble des gènes) de P. falciparum a conduit au développement de nouvelles .études à large échelle dans le domaine des protéines du parasite (protéome) ; dans l'utilisation d'algorithmes, de techniques informatiques et statistiques pour l'analyse de données biologiques (bioinformatique) et dans les technologies de transcription et de profiles d'expression (transcriptome). Nous avons identifié, en utilisant les outils ci-dessus, des nouvelles protéines antigéniques qui sont présentes au stade sanguin de la malaria. Nous avons sélectionné, parmi ces protéines, celles contenant un motif dit "a-helical coiled-coil" qui sont des domaines impliqués dans un large éventail de fonctions biologiques. Des peptides représentant ces régions structurellement stables ont été synthétisés et utilisés pour immuniser des souris. La plupart des peptides testés sont immunogéniques et induisent un titre variable d'anticorps déterminé par ELISA. Les résultats de tests d'immunofluorescence indiquent que plusieurs sera de souris immunisées avec ces peptides reconnaissent les protéines natives exprimées à la surface des globules rouges infectés. En parallèle, des études d'ADCI in vitro montrent que des anticorps humains purifiés à partir de ces peptides en présence de monocytes inhibent la croissance du parasite de manière similaire à celle observée chez des adultes africains protégés. Des études d'antigénicité utilisant des sera de personnes immunes de différents âges (adultes et enfants) vivant dans des régions endémiques montrent que la majorité des peptides sont reconnus avec une haute prévalence. 73 peptides ont été testés dans des études épidémiologiques dans 2 villages côtiers du Kenya Ces 2 groupes vivent dans des zones bien distinctes et les prélèvements n'ont pas été effectués pendant la même période. Dans ces études, la réponse anticorps dirigée contre les peptides synthétiques a été testée en utilisant 467 échantillons sanguins d'enfants à risque de développer un épisode de malaria afin de caractériser le niveau de reconnaissance des peptides en fonction de l'âge et de déterminer le rôle des anticorps anti-peptides dans la protection contre la malaria cérébrale. Parmi ces peptides, 10 sont associés à une protection contre un épisode de malaria dans le premier village alors qu'un seul l'est dans les 2 villages. Ces résultats sont en faveur de futures études qui évalueraient systématiquement l'immunogénicité des peptides intéressants dans le but d'établir des critères de sélection clairs pour le développement d'un vaccin.

Stereotactic body radiation therapy in stage I inoperable lung cancer: from palliative to curative options.

Surgery has historically been the standard of care for operable stage I non-small cell lung cancer (NSCLC). However, nearly one-quarter of patients with stage I NSCLC will not undergo surgery because of medical comorbidity or other factors. Stereotactic ablative radiotherapy (SABR) is the new standard of care for these patients. SABR offers high local tumour control rates rivalling the historical results of surgery and is generally well tolerated by patients with both peripheral and centrally located tumours. This article reviews the history of SABR for stage I NSCLC, summarises the currently available data on efficacy and toxicity, and describes some of the currently controversial aspects of this treatment.

Distinct profiles of cytotoxic granules in memory CD8 T cells correlate with function, differentiation stage, and antigen exposure.

Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules. Degranulation activity and cytotoxic granules (perforin plus granzymes) generally define CD8 T cells with cytotoxic function. In this study, we have investigated the expression of granzyme K (GrmK) in comparison to that of GrmA, GrmB, and perforin. The expression of the cytotoxic granules was assessed in virus-specific CD8 T cells specific to influenza virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), or human immunodeficiency virus type 1 (HIV-1). We observed a dichotomy between GrmK and perforin expression in virus-specific CD8 T cells. The profile in influenza virus-specific CD8 T cells was perforin(-) GrmB(-) GrmA(+/-) GrmK(+); in CMV-specific cells, it was perforin(+) GrmB(+) GrmA(+) GrmK(-/+); and in EBV- and HIV-1-specific cells, it was perforin(-/+) GrmB(+) GrmA(+) GrmK(+). On the basis of the delineation of memory and effector CD8 T cells with CD45RA and CD127, the GrmK(+) profile was associated with early-stage memory CD8 T-cell differentiation, the perforin(+) GrmB(+) GrmA(+) profile with advanced-stage differentiation, and the GrmB(+) GrmA(+) Grmk(+) profile with intermediate-stage differentiation. Furthermore, perforin and GrmB but not GrmA and GrmK correlated with cytotoxic activity. Finally, changes in antigen exposure in vitro and in vivo during primary HIV-1 infection and vaccination modulated cytotoxic granule profiles. These results advance our understanding of the relationship between distinct profiles of cytotoxic granules in memory CD8 T cells and function, differentiation stage, and antigen exposure.

Biometal muscle to restore atrial transport function in a permanent atrial fibrillation animal model: a potential tool in the treatment of end-stage heart failure.

BACKGROUND: Half of the patients with end-stage heart failure suffer from persistent atrial fibrillation (AF). Atrial kick (AK) accounts for 10-15% of the ejection fraction. A device restoring AK should significantly improve cardiac output (CO) and possibly delay ventricular assist device (VAD) implantation. This study has been designed to assess the mechanical effects of a motorless pump on the right chambers of the heart in an animal model. METHODS: Atripump is a dome-shaped biometal actuator electrically driven by a pacemaker-like control unit. In eight sheep, the device was sutured onto the right atrium (RA). AF was simulated with rapid atrial pacing. RA ejection fraction (EF) was assessed with intracardiac ultrasound (ICUS) in baseline, AF and assisted-AF status. In two animals, the pump was left in place for 4 weeks and then explanted. Histology examination was carried out. The mean values for single measurement per animal with +/-SD were analysed. RESULTS: The contraction rate of the device was 60 per min. RA EF was 41% in baseline, 7% in AF and 21% in assisted-AF conditions. CO was 7+/-0.5 l min(-1) in baseline, 6.2+/-0.5 l min(-1) in AF and 6.7+/-0.5 l min(-1) in assisted-AF status (p<0.01). Histology of the atrium in the chronic group showed chronic tissue inflammation and no sign of tissue necrosis. CONCLUSIONS: The artificial muscle restores the AK and improves CO. In patients with end-stage cardiac failure and permanent AF, if implanted on both sides, it would improve CO and possibly delay or even avoid complex surgical treatment such as VAD implantation.

Ultra-high pressure liquid chromatography-mass spectrometry for plant metabolomics: a systematic comparison of high-resolution quadrupole-time-of-flight and single stage Orbitrap mass spectrometers.

The response of Arabidopsis to stress caused by mechanical wounding was chosen as a model to compare the performances of high resolution quadrupole-time-of-flight (Q-TOF) and single stage Orbitrap (Exactive Plus) mass spectrometers in untargeted metabolomics. Both instruments were coupled to ultra-high pressure liquid chromatography (UHPLC) systems set under identical conditions. The experiment was divided in two steps: the first analyses involved sixteen unwounded plants, half of which were spiked with pure standards that are not present in Arabidopsis. The second analyses compared the metabolomes of mechanically wounded plants to unwounded plants. Data from both systems were extracted using the same feature detection software and submitted to unsupervised and supervised multivariate analysis methods. Both mass spectrometers were compared in terms of number and identity of detected features, capacity to discriminate between samples, repeatability and sensitivity. Although analytical variability was lower for the UHPLC-Q-TOF, generally the results for the two detectors were quite similar, both of them proving to be highly efficient at detecting even subtle differences between plant groups. Overall, sensitivity was found to be comparable, although the Exactive Plus Orbitrap provided slightly lower detection limits for specific compounds. Finally, to evaluate the potential of the two mass spectrometers for the identification of unknown markers, mass and spectral accuracies were calculated on selected identified compounds. While both instruments showed excellent mass accuracy (<2.5ppm for all measured compounds), better spectral accuracy was recorded on the Q-TOF. Taken together, our results demonstrate that comparable performances can be obtained at acquisition frequencies compatible with UHPLC on Q-TOF and Exactive Plus MS, which may thus be equivalently used for plant metabolomics.

Ant queens (Hymenoptera: Formicidae) are attracted to fungal pathogens during the initial stage of colony founding

Ant queens that attempt to disperse and found new colonies independently face high mortality risks. The exposure of queens to soil entomopathogens during claustral colony founding may be particularly harmful, as founding queens lack the protection conferred by mature colonies. Here, we tested the hypotheses that founding queens (I) detect and avoid nest sites that are contaminated by fungal pathogens, and (II) tend to associate with other queens to benefit from social immunity when nest sites are contaminated. Surprisingly, in nest choice assays, young Formica selysi BONDROIT, 1918 queens had an initial preference for nest sites contaminated by two common soil entomopathogenic fungi, Beauveria bassiana and Metarhizium brunneum. Founding queens showed a similar preference for the related but non-entomopathogenic fungus Fusarium graminearum. In contrast, founding queens had no significant preference for the more distantly related nonentomopathogenic fungus Petromyces alliaceus, nor for heat-killed spores of B. bassiana. Finally, founding queens did not increase the rate of queen association in presence of B. bassiana. The surprising preference of founding queens for nest sites contaminated by live entomopathogenic fungi suggests that parasites manipulate their hosts or that the presence of specific fungi is a cue associated with suitable nesting sites.

Decreased local control following radiation therapy alone in early-stage glottic carcinoma with anterior commissure extension.

PURPOSE: To assess the patterns of failure in the treatment of early-stage squamous cell carcinoma of the glottic larynx. PATIENTS AND METHODS: Between 1983-2000, 122 consecutive patients treated for early laryngeal cancer (UICC T1N0 and T2N0) by radical radiation therapy (RT) were retrospectively studied. Male-to-female ratio was 106 : 16, and median age 62 years (35-92 years). There were 68 patients with T1a, 18 with T1b, and 36 with T2 tumors. Diagnosis was made by biopsy in 104 patients, and by laser vaporization or stripping in 18. Treatment planning consisted of three-dimensional (3-D) conformal RT in 49 (40%) patients including nine patients irradiated using arytenoid protection. A median dose of 70 Gy (60-74 Gy) was given (2 Gy/fraction) over a median period of 46 days (21-79 days). Median follow-up period was 85 months. RESULTS: The 5-year overall, cancer-specific, and disease-free survival amounted to 80%, 94%, and 70%, respectively. 5-year local control was 83%. Median time to local recurrence in 19 patients was 13 months (5-58 months). Salvage treatment consisted of surgery in 17 patients (one patient refused salvage and one was inoperable; total laryngectomy in eleven, and partial laryngectomy or cordectomy in six patients). Six patients died because of laryngeal cancer. Univariate analyses revealed that prognostic factors negatively influencing local control were anterior commissure extension, arytenoid protection, and total RT dose < 66 Gy. Among the factors analyzed, multivariate analysis (Cox model) demonstrated that anterior commissure extension, arytenoid protection, and male gender were the worst independent prognostic factors in terms of local control. CONCLUSION: For early-stage laryngeal cancer, outcome after RT is excellent. In case of anterior commissure extension, surgery or higher RT doses are warranted. Because of a high relapse risk, arytenoid protection should not be attempted.

Toxicity at three years with and without irradiation of the internal mammary and medial supraclavicular lymph node chain in stage I to III breast cancer (EORTC trial 22922/10925).

INTRODUCTION: The EORTC 22922/10925 trial investigated the potential survival benefit and toxicity of elective irradiation of the internal mammary and medial supraclavicular (IM-MS) nodes Accrual completed in January 2004 and first results are expected in 2012. We present the toxicity reported until year 3 after treatment. PATIENTS AND METHODS: At each visit, toxicity was reported but severity was not graded routinely. Toxicity rates and performance status (PS) changes at three years were compared by chi(2) tests and logistic regression models in all the 3,866 of 4,004 patients eligible to the trial who received the allocated treatment. RESULTS: Only lung (fibrosis; dyspnoea; pneumonitis; any lung toxicities) (4.3% vs. 1.3%; p < 0.0001) but not cardiac toxicity (0.3% vs. 0.4%; p = 0.55) significantly increased with IM-MS treatment. No significant worsening of the PS was observed (p = 0.79), suggesting that treatment-related toxicity does not impair patient's daily activities. CONCLUSIONS: IM-MS irradiation seems well tolerated and does not significantly impair WHO PS at three years. A follow-up period of at least 10 years is needed to determine whether cardiac toxicity is increased after radiotherapy.

The European Respiratory Society spirometry tent: a unique form of screening for airway obstruction.

In order to raise public awareness of the importance of early detection of airway obstruction and to enable many people who had not been tested previously to have their lung function measured, the European Lung Foundation and the European Respiratory Society (ERS) organised a spirometry testing tent during the annual ERS Congresses in 2004-2009. Spirometry was performed during the ERS Congresses in volunteers; all participants answered a simple, brief questionnaire on their descriptive characteristics, smoking and asthma. Portable spirometers were freely provided by the manufacturer. Nurses and doctors from pulmonary departments of local hospitals/universities gave their service for free. Lower limit of normal (LLN) and Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria for diagnosing and grading airway obstruction were used. Of 12,448 participants in six congress cities, 10,395 (83.5%) performed acceptable spirometry (mean age 51.0 ± 18.4 yrs; 25.5% smokers; 5.5% asthmatic). Airway obstruction was present in 12.4% of investigated subjects according to LLN criteria and 20.3% according to GOLD criteria. Through multinomial logistic regression analysis, age, smoking habits and asthma were significant risk factors for airway obstruction. Relative risk ratio and 95% confidence interval for LLN stage I, for example, was 2.9 (2.0-4.1) for the youngest age (≤ 19 yrs), 1.9 (1.2-3.0) for the oldest age (≥ 80 yrs), 2.4 (2.0-2.9) for current smokers and 2.8 (2.2-3.6) for reported asthma diagnosis. In addition to being a useful advocacy tool, the spirometry tent represents an unusual occasion for early detection of airway obstruction in large numbers of city residents with an important public health perspective.

A deterministic model to assess the impact of AIDS interventions in countries with generalized HIV epidemic. An application to Botswana

General introductionThe Human Immunodeficiency/Acquired Immunodeficiency Syndrome (HIV/AIDS) epidemic, despite recent encouraging announcements by the World Health Organization (WHO) is still today one of the world's major health care challenges.The present work lies in the field of health care management, in particular, we aim to evaluate the behavioural and non-behavioural interventions against HIV/AIDS in developing countries through a deterministic simulation model, both in human and economic terms. We will focus on assessing the effectiveness of the antiretroviral therapies (ART) in heterosexual populations living in lesser developed countries where the epidemic has generalized (formerly defined by the WHO as type II countries). The model is calibrated using Botswana as a case study, however our model can be adapted to other countries with similar transmission dynamics.The first part of this thesis consists of reviewing the main mathematical concepts describing the transmission of infectious agents in general but with a focus on human immunodeficiency virus (HIV) transmission. We also review deterministic models assessing HIV interventions with a focus on models aimed at African countries. This review helps us to recognize the need for a generic model and allows us to define a typical structure of such a generic deterministic model.The second part describes the main feed-back loops underlying the dynamics of HIV transmission. These loops represent the foundation of our model. This part also provides a detailed description of the model, including the various infected and non-infected population groups, the type of sexual relationships, the infection matrices, important factors impacting HIV transmission such as condom use, other sexually transmitted diseases (STD) and male circumcision. We also included in the model a dynamic life expectancy calculator which, to our knowledge, is a unique feature allowing more realistic cost-efficiency calculations. Various intervention scenarios are evaluated using the model, each of them including ART in combination with other interventions, namely: circumcision, campaigns aimed at behavioral change (Abstain, Be faithful or use Condoms also named ABC campaigns), and treatment of other STD. A cost efficiency analysis (CEA) is performed for each scenario. The CEA consists of measuring the cost per disability-adjusted life year (DALY) averted. This part also describes the model calibration and validation, including a sensitivity analysis.The third part reports the results and discusses the model limitations. In particular, we argue that the combination of ART and ABC campaigns and ART and treatment of other STDs are the most cost-efficient interventions through 2020. The main model limitations include modeling the complexity of sexual relationships, omission of international migration and ignoring variability in infectiousness according to the AIDS stage.The fourth part reviews the major contributions of the thesis and discusses model generalizability and flexibility. Finally, we conclude that by selecting the adequate interventions mix, policy makers can significantly reduce the adult prevalence in Botswana in the coming twenty years providing the country and its donors can bear the cost involved.Part I: Context and literature reviewIn this section, after a brief introduction to the general literature we focus in section two on the key mathematical concepts describing the transmission of infectious agents in general with a focus on HIV transmission. Section three provides a description of HIV policy models, with a focus on deterministic models. This leads us in section four to envision the need for a generic deterministic HIV policy model and briefly describe the structure of such a generic model applicable to countries with generalized HIV/AIDS epidemic, also defined as pattern II countries by the WHO.