Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Bulk-up synchronization of successive larval cohorts of Anopheles gambiae and Anopheles coluzzii through temperature reduction at early larval stages: effect on emergence rate, body size and mating success.

BACKGROUND: Malaria persists as a huge medical and economic burden. Although the number of cases and death rates have reduced in recent years, novel interventions are a necessity if such gains are to be maintained. Alternative methods to target mosquito vector populations that involve the release of large numbers genetically modified mosquitoes are in development. However, their successful introduction will require innovative strategies to bulk-up mosquito numbers and improve mass rearing protocols for Anopheles mosquitoes. METHODS: The relationship between mosquito aquatic stage development and temperature was exploited so that multiple cohorts of mosquitoes, from separate egg batches, could be synchronized to 'bulk-up' the number of mosquitoes released. First instar larvae were separated into two cohorts: the first, maintained under standard insectary conditions at 27oC, the second subjected to an initial 5-day cooling period at 19oC. RESULTS: Cooling of 1st instars slowed the mean emergence times of Anopheles coluzzii and Anopheles gambiae by 2.4 and 3.5 days, respectively, compared to their 27oC counterparts. Pupation and emergence rates were good (> 85 %) in all conditions. Temperature adjustment had no effect on mosquito sex ratio and adult fitness parameters such as body size and mating success. CONCLUSIONS: Bulk-up larval synchronization is a simple method allowing more operational flexibility in mosquito production towards mark-release-recapture studies and mass release interventions.

Lead Optimization of Dehydroemetine for Repositioned Use in Malaria.

Drug repositioning offers an effective alternative to de novo drug design to tackle the urgent need for novel antimalarial treatments. The antiamoebic compound emetine dihydrochloride has been identified as a potent in vitro inhibitor of the multidrug-resistant strain K1 of Plasmodium falciparum (50% inhibitory concentration [IC50], 47 nM ± 2.1 nM [mean ± standard deviation]). Dehydroemetine, a synthetic analogue of emetine dihydrochloride, has been reported to have less-cardiotoxic effects than emetine. The structures of two diastereomers of dehydroemetine were modeled on the published emetine binding site on the cryo-electron microscopy (cryo-EM) structure with PDB code 3J7A (P. falciparum 80S ribosome in complex with emetine), and it was found that (-)-R,S-dehydroemetine mimicked the bound pose of emetine more closely than did (-)-S,S-dehydroisoemetine. (-)-R,S-dehydroemetine (IC50 71.03 ± 6.1 nM) was also found to be highly potent against the multidrug-resistant K1 strain of P. falciparum compared with (-)-S,S-dehydroisoemetine (IC50, 2.07 ± 0.26 µM), which loses its potency due to the change of configuration at C-1'. In addition to its effect on the asexual erythrocytic stages of P. falciparum, the compound exhibited gametocidal properties with no cross-resistance against any of the multidrug-resistant strains tested. Drug interaction studies showed (-)-R,S-dehydroemetine to have synergistic antimalarial activity with atovaquone and proguanil. Emetine dihydrochloride and (-)-R,S-dehydroemetine failed to show any inhibition of the hERG potassium channel and displayed activity affecting the mitochondrial membrane potential, indicating a possible multimodal mechanism of action.

Drug repositioning as a route to anti-malarial drug discovery: preliminary investigation of the in vitro anti-malarial efficacy of emetine dihydrochloride hydrate.

BACKGROUND: Drug repurposing or repositioning refers to the usage of existing drugs in diseases other than those it was originally used for. For diseases like malaria, where there is an urgent need for active drug candidates, the strategy offers a route to significantly shorten the traditional drug development pipelines. Preliminary high-throughput screens on patent expired drug libraries have recently been carried out for Plasmodium falciparum. This study reports the systematic and objective further interrogation of selected compounds reported in these studies, to enable their repositioning as novel stand-alone anti-malarials or as combinatorial partners. METHODS: SYBR Green flow cytometry and micro-titre plate assays optimized in the laboratory were used to monitor drug susceptibility of in vitro cultures of P. falciparum K1 parasite strains. Previously described fixed-ratio methods were adopted to investigate drug interactions. RESULTS: Emetine dihydrochloride hydrate, an anti-protozoal drug previously used for intestinal and tissue amoebiasis was shown to have potent inhibitory properties (IC50 doses of ~ 47 nM) in the multidrug resistant K1 strain of P. falciparum. The sum 50% fractional inhibitory concentration (∑FIC50, 90) of the interaction of emetine dihydrochloride hydrate and dihydroartemisinin against the K1 strains of P. falciparum ranged from 0.88-1.48. CONCLUSION: The results warrant further investigation of emetine dihydrochloride hydrate as a potential stand-alone anti-malarial option. The interaction between the drug and the current front line dihydroartemisinin ranged from additive to mildly antagonistic in the fixed drug ratios tested.

Variation in apoptosis mechanisms employed by malaria parasites: the roles of inducers, dose dependence and parasite stages.

BACKGROUND: Plasmodium berghei ookinetes exhibit an apoptotic phenotype when developing within the mosquito midgut lumen or when cultured in vitro. Markers of apoptosis increase when they are exposed to nitric oxide or reactive oxygen species but high concentrations of hydrogen peroxide cause death without observable signs of apoptosis. Chloroquine and other drugs have been used to induce apoptosis in erythrocytic stages of Plasmodium falciparum and to formulate a putative pathway involving cysteine protease activation and mitochondrial membrane permeabilization; initiated, at least in the case of chloroquine, after its accumulation in the digestive vacuole causes leakage of the vacuole contents. The lack of a digestive vacuole in ookinetes prompted the investigation of the effect of chloroquine and staurosporine on this stage of the life cycle. Finally, the suggestion that apoptosis may have evolved as a strategy employed by ookinetes to increase the fitness of surviving parasites was explored by determining whether increasing the ecological triggers parasite density and nutrient depletion induced apoptosis. METHODS: Ookinetes were grown in culture then either exposed to hydrogen peroxide, chloroquine or staurosporine, or incubated at different densities and in different media. The proportion of ookinetes displaying positive markers for apoptosis in treated samples was compared with controls and results were analyzed using analysis of variance followed by a Turkey's test, or a Kruskal-Wallis test as appropriate. RESULTS: Hydrogen peroxide below 50 µM triggered apoptosis but cell membranes were rapidly compromised by higher concentrations, and the mode of death could not be defined. Both chloroquine and staurosporine cause a significant increase in ookinetes with condensed chromatin, caspase-like activity and, in the case of chloroquine, phosphatidylserine translocation and DNA fragmentation (not investigated for staurosporine). However, mitochondrial membrane potential remained intact. No relationship between ookinete density and apoptosis was detected but nutrient depletion significantly increased the proportion of ookinetes with chromatin condensation in four hours. CONCLUSIONS: It is proposed that both a mitochondrial and an amitochondrial apoptotic pathway may be involved, dependent upon the trigger that induces apoptosis, and that pathways may differ between erythrocytic stages and ookinetes, or between rodent and human malaria parasites.

Elizabethkingia anophelis sp. nov., isolated from the midgut of the mosquito Anopheles gambiae.

The taxonomic position, growth characteristics and antibiotic resistance properties of a slightly yellow-pigmented bacterial strain, designated R26(T), isolated from the midgut of the mosquito Anopheles gambiae, were studied. The isolate produced rod-shaped cells, which stained Gram-negative. The bacterium had two growth optima at 30-31 °C and 37 °C. Strain R26(T) demonstrated natural antibiotic resistance to ampicillin, chloramphenicol, kanamycin, streptomycin and tetracycline. 16S rRNA gene sequence analysis revealed that the isolate showed 98.6 % sequence similarity to that of Elizabethkingia meningoseptica ATCC 13253(T) and 98.2 % similarity to that of Elizabethkingia miricola GTC 862(T). The major fatty acids of strain R26(T) were iso-C(15 : 0), iso-C(17 : 0) 3-OH and summed feature 4 (iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c/t). Strain R26(T) contained only menaquinone MK-6 and showed a complex polar lipid profile consisting of diphosphatidylglycerol, phosphatidylinositol, an unknown phospholipid and unknown polar lipids and glycolipids. DNA-DNA hybridization experiments with E. meningoseptica CCUG 214(T) ( = ATCC 13253(T)) and E. miricola KCTC 12492(T) ( = GTC 862(T)) gave relatedness values of 34.5 % (reciprocal 41.5 %) and 35.0 % (reciprocal 25.7 %), respectively. DNA-DNA hybridization results and some differentiating biochemical properties indicate that strain R26(T) represents a novel species, for which the name Elizabethkingia anophelis sp. nov. is proposed. The type strain is R26(T) ( = CCUG 60038(T) = CCM 7804(T)).

Unexpected encounter of the parasitic kind.

Both parasitology and stem cell research are important disciplines in their own right. Parasites are a real threat to human health causing a broad spectrum of diseases and significant annual rates morbidity and mortality globally. Stem cell research, on the other hand, focuses on the potential for regenerative medicine for a range of diseases including cancer and regenerative therapies. Though these two topics might appear distant, there are some "unexpected encounters". In this review, we summarise the various links between parasites and stem cells. First, we discuss how parasites' own stem cells represent interesting models of regeneration that can be translated to human stem cell regeneration. Second, we explore the interactions between parasites and host stem cells during the course of infection. Third, we investigate from a clinical perspective, how stem cell regeneration can be exploited to help circumvent the damage induced by parasitic infection and its potential to serve as treatment options for parasitic diseases in the future. Finally, we discuss the importance of screening for pathogens during organ transplantation by presenting some clinical cases of parasitic infection following stem cell therapy.

A comparative evaluation of arterial blood flow and the healing response after femoral artery closure using angio-seal STS Plus and StarClose in a porcine model.

OBJECTIVES: This study prospectively evaluated the acute and chronic arterial blood flow and vascular pathology after vessel closure using two commonly used closure devices controlled by deploying both devices in each animal. BACKGROUND: Several vessel closure systems are approved for clinical use; however, few direct comparisons have ever been performed and no randomized case controlled study has been published using FDA-approved devices. METHODS: Nineteen Sous Scroufulae pigs underwent bilateral percutaneous arteriotomies using ultrasound-guided 6 Fr sheath insertion in both common femoral arteries. The femoral access site was then closed using either an Angio-Seal STS Plus, an absorbable collagen sponge, or StarClose, a self-closing nitinol clip. Angiograms and ultrasound of the site were performed prior to closure and immediately afterwards. At follow-up, ultrasound was performed at the site and the specimens were sent for histopathology. RESULTS: Baseline femoral artery diameters (centimeters) were similar in both groups by U/S (5.2 +/- 0.3, 5.3 +/- 0.3) and quantitative angiography (4.6 +/- 0.7, 4.6 +/- 0.8). Postdeployment angiograms showed a vessel diameter stenosis of 65%+/- 24% with Angio-Seal (n = 18) and 50%+/- 22% with StarClose (n = 18), P = 0.04. 2D U/S performed immediately postdeployment showed vessel diameter stenosis of 59%+/- 33.0 with Angio-Seal (n = 19), and 35%+/- 20 with StarClose (n = 19), P = 0.01. At 7-, 30-, and 60-day follow-up, no appreciable differences in the vessel diameter were observed by U/S. At early follow-up (7 and 30 days), Angio-Seal arteriotomy closure sites were associated with higher inflammatory and hemorrhage scores, but no difference was seen at late (60-day) follow-up. CONCLUSIONS: The StarClose closure device is associated with less short-term vessel injury compared to Angio-Seal STS Plus; however, this difference was not statistically significant after 60 days.

Checks and balances? DNA replication and the cell cycle in Plasmodium.

It is over 100 years since the life-cycle of the malaria parasite Plasmodium was discovered, yet its intricacies remain incompletely understood - a knowledge gap that may prove crucial for our efforts to control the disease. Phenotypic screens have partially filled the void in the antimalarial drug market, but as compound libraries eventually become exhausted, new medicines will only come from directed drug development based on a better understanding of fundamental parasite biology. This review focusses on the unusual cell cycles of Plasmodium, which may present a rich source of novel drug targets as well as a topic of fundamental biological interest. Plasmodium does not grow by conventional binary fission, but rather by several syncytial modes of replication including schizogony and sporogony. Here, we collate what is known about the various cell cycle events and their regulators throughout the Plasmodium life-cycle, highlighting the differences between Plasmodium, model organisms and other apicomplexan parasites and identifying areas where further study is required. The possibility of DNA replication and the cell cycle as a drug target is also explored. Finally the use of existing tools, emerging technologies, their limitations and future directions to elucidate the peculiarities of the Plasmodium cell cycle are discussed.

Development of a Photo-Cross-Linkable Diaminoquinazoline Inhibitor for Target Identification in Plasmodium falciparum.

Diaminoquinazolines represent a privileged scaffold for antimalarial discovery, including use as putative Plasmodium histone lysine methyltransferase inhibitors. Despite this, robust evidence for their molecular targets is lacking. Here we report the design and development of a small-molecule photo-cross-linkable probe to investigate the targets of our diaminoquinazoline series. We demonstrate the effectiveness of our designed probe for photoaffinity labeling of Plasmodium lysates and identify similarities between the target profiles of the probe and the representative diaminoquinazoline BIX-01294. Initial pull-down proteomics experiments identified 104 proteins from different classes, many of which are essential, highlighting the suitability of the developed probe as a valuable tool for target identification in Plasmodium falciparum.

A high throughput screen for next-generation leads targeting malaria parasite transmission.

Spread of parasite resistance to artemisinin threatens current frontline antimalarial therapies, highlighting the need for new drugs with alternative modes of action. Since only 0.2-1% of asexual parasites differentiate into sexual, transmission-competent forms, targeting this natural bottleneck provides a tangible route to interrupt disease transmission and mitigate resistance selection. Here we present a high-throughput screen of gametogenesis against a ~70,000 compound diversity library, identifying seventeen drug-like molecules that target transmission. Hit molecules possess varied activity profiles including male-specific, dual acting male-female and dual-asexual-sexual, with one promising N-((4-hydroxychroman-4-yl)methyl)-sulphonamide scaffold found to have sub-micromolar activity in vitro and in vivo efficacy. Development of leads with modes of action focussed on the sexual stages of malaria parasite development provide a previously unexplored base from which future therapeutics can be developed, capable of preventing parasite transmission through the population.

Immunogenicity of Oka/Merck varicella vaccine in children vaccinated at 12-14 months of age versus 15-23 months of age.

OBJECTIVE: Recent reports suggest that breakthrough varicella may be more common in children when the Oka/Merck varicella vaccine is given at 12-14 months of age than when it is given at older ages. An analysis of 5 postlicensure clinical trials with this vaccine was conducted to evaluate immune response relative to the age of the vaccine recipient. METHODS: In 5 clinical trials, 3771 children, 12 through 23 months of age with no history of varicella, received an injection of varicella vaccine. Varicella-zoster virus (VZV) antibody was measured 6 weeks postvaccination by glycoprotein enzyme-linked immunosorbent assay (gpELISA), an assay that correlates with neutralizing antibody. Endpoints evaluated were the response rate (percent of subjects with VZV antibody > or =5 gpELISA units/mL, a titer shown to correlate with protection) and geometric mean titer (GMT) of VZV antibody. Each endpoint was compared across 3 age groups (12-14, 15-17, and 18-23 months of age). Response rates by initial VZV serostatus were evaluated for children vaccinated at 12-14 months of age to assess whether maternal antibody had an impact on the immune response. RESULTS: The response rates were similar among 12-14, 15-17, and 18-23 month olds (93.8, 90.8, and 93.1%, respectively); GMTs were significantly higher among the 12-14 month olds (15.1, 13.5, and 13.7 gpELISA units/mL, respectively). Among children 12-14 months of age, response rates and GMTs were similar regardless of their prevaccination VZV serostatus. CONCLUSIONS: Oka/Merck varicella vaccine is highly immunogenic when given to children 12-14 months of age. The immunogenicity profile is similar to that of children 15-17 and 18-23 months of age. The presence of low titers of VZV antibody before vaccination did not influence vaccine response in 12-14 month olds. These results support current recommendations for universal varicella vaccination beginning at 12 months of age.

Investigating antimalarial drug interactions of emetine dihydrochloride hydrate using CalcuSyn-based interactivity calculations.

The widespread introduction of artemisinin-based combination therapy has contributed to recent reductions in malaria mortality. Combination therapies have a range of advantages, including synergism, toxicity reduction, and delaying the onset of resistance acquisition. Unfortunately, antimalarial combination therapy is limited by the depleting repertoire of effective drugs with distinct target pathways. To fast-track antimalarial drug discovery, we have previously employed drug-repositioning to identify the anti-amoebic drug, emetine dihydrochloride hydrate, as a potential candidate for repositioned use against malaria. Despite its 1000-fold increase in in vitro antimalarial potency (ED50 47 nM) compared with its anti-amoebic potency (ED50 26-32 uM), practical use of the compound has been limited by dose-dependent toxicity (emesis and cardiotoxicity). Identification of a synergistic partner drug would present an opportunity for dose-reduction, thus increasing the therapeutic window. The lack of reliable and standardised methodology to enable the in vitro definition of synergistic potential for antimalarials is a major drawback. Here we use isobologram and combination-index data generated by CalcuSyn software analyses (Biosoft v2.1) to define drug interactivity in an objective, automated manner. The method, based on the median effect principle proposed by Chou and Talalay, was initially validated for antimalarial application using the known synergistic combination (atovaquone-proguanil). The combination was used to further understand the relationship between SYBR Green viability and cytocidal versus cytostatic effects of drugs at higher levels of inhibition. We report here the use of the optimised Chou Talalay method to define synergistic antimalarial drug interactivity between emetine dihydrochloride hydrate and atovaquone. The novel findings present a potential route to harness the nanomolar antimalarial efficacy of this affordable natural product.

"Omics"-Informed Drug and Biomarker Discovery: Opportunities, Challenges and Future Perspectives.

The pharmaceutical industry faces unsustainable program failure despite significant increases in investment. Dwindling discovery pipelines, rapidly expanding R&D budgets and increasing regulatory control, predict significant gaps in the future drug markets. The cumulative duration of discovery from concept to commercialisation is unacceptably lengthy, and adds to the deepening crisis. Existing animal models predicting clinical translations are simplistic, highly reductionist and, therefore, not fit for purpose. The catastrophic consequences of ever-increasing attrition rates are most likely to be felt in the developing world, where resistance acquisition by killer diseases like malaria, tuberculosis and HIV have paced far ahead of new drug discovery. The coming of age of Omics-based applications makes available a formidable technological resource to further expand our knowledge of the complexities of human disease. The standardisation, analysis and comprehensive collation of the "data-heavy" outputs of these sciences are indeed challenging. A renewed focus on increasing reproducibility by understanding inherent biological, methodological, technical and analytical variables is crucial if reliable and useful inferences with potential for translation are to be achieved. The individual Omics sciences-genomics, transcriptomics, proteomics and metabolomics-have the singular advantage of being complimentary for cross validation, and together could potentially enable a much-needed systems biology perspective of the perturbations underlying disease processes. If current adverse trends are to be reversed, it is imperative that a shift in the R&D focus from speed to quality is achieved. In this review, we discuss the potential implications of recent Omics-based advances for the drug development process.

Ten year follow-up of healthy children who received one or two injections of varicella vaccine.

BACKGROUND: The rate of varicella and persistence of varicella antibody after a one dose vs. a two dose regimen of varicella virus vaccine live Oka/Merck (VARIVAX; Merck & Co., Inc., West Point, PA) in approximately 2000 children were compared during a 9- to 10-year follow-up period. METHODS: Children 12 months to 12 years of age with a negative history of varicella were randomized in late 1991 to early 1993 to receive either one or two injections of varicella vaccine given 3 months apart. Subjects were actively followed for varicella, any varicella-like illness or zoster and any exposures to varicella or zoster on a yearly basis for 10 years after vaccination. Persistence of varicella antibody was measured yearly for 9 years. RESULTS: Most cases of varicella reported in recipients of one or two injections of vaccine were mild. The risk of developing varicella >42 days postvaccination during the 10-year observation period was 3.3-fold lower (P < 0.001) in children who received two injections than in those who received one injection (2.2% vs. 7.3%, respectively). The estimated vaccine efficacy for the 10-year observation period was 94.4% for one injection and 98.3% for two injections (P < 0.001). Measurable serum antibody persisted for 9 years in all subjects. CONCLUSIONS: Administration of either one or two injections of varicella vaccine to healthy children results in long term protection against most varicella disease. The two dose regimen was significantly more effective than a single injection.

Inverse relationship between six week postvaccination varicella antibody response to vaccine and likelihood of long term breakthrough infection.

BACKGROUND: We used the large clinical database that supported the development of Oka/ Merck varicella vaccine to study the relationship between the primary varicella antibody response, as determined by gpELISA, an enzyme-linked immunosorbent assay that detects antibodies to varicella-zoster virus (VZV) glycoprotein, and the subsequent risk of postvaccination breakthrough varicella. METHODS: We vaccinated 1,164 healthy children with a single dose of varicella vaccine containing 2900 to 9000 plaque-forming units/dose. The primary immune response to vaccination was determined by gpELISA 6 weeks after vaccination. Subjects were followed annually for 7 years to ascertain cases of breakthrough varicella. RESULTS: The estimated vaccine efficacy among children with a 6-week postvaccination antibody titer of > or = 5 gpELISA units was 95.5% (95% confidence interval, 94.2%, 96.8%) compared with 83.5% (95% confidence interval, 76.9%, 89.5%) for subjects with a titer of <5 gpELISA units. Children with a 6-week postvaccination antibody titer of <5 gpELISA units were 3.5 times more likely than those with a titer of > or = 5 gpELISA units to develop breakthrough varicella. CONCLUSIONS: We identified a 6-week postvaccination antibody titer of > or = 5 gpELISA units as an approximate correlate of protection. In addition we established an accelerated failure time model based on log normal hazard that predicted varicella breakthrough rates based on the distribution of 6-week postvaccination varicella antibody titers.

Vaccination with measles, mumps and rubella vaccine and varicella vaccine: safety, tolerability, immunogenicity, persistence of antibody and duration of protection against varicella in healthy children.

BACKGROUND: Administration of M-M-R II (Measles, Mumps and Rubella Virus Vaccine, Live) and VARIVAX [Varicella Virus Vaccine Live (Oka/Merck)] given concomitantly at separate injection sites during the same office visit could increase vaccine compliance by reducing the number of health care visits for immunizations. We compared the safety and immunogenicity of M-M-R II and VARIVAX given concomitantly at separate sites (Group A) with administration of the two vaccines 6 weeks apart (Group B) as well as the persistence of varicella antibody and the duration of protection afforded by varicella vaccine. METHODS: A total of 603 healthy children, ages 12 months to 6 years, with no history of measles, mumps, rubella, varicella and zoster or vaccination against these diseases, were randomized to either Group A or B and were followed for clinical reactions and serologic responses to all four viral components. Children were enrolled from August through December, 1993. Subjects were followed for 5 years to evaluate persistence of varicella antibody and breakthrough varicella rates. We compared breakthrough rates to expected attack rates in unvaccinated children to produce estimates of vaccine efficacy. RESULTS: Both vaccine regimens were generally well-tolerated. There were no significant differences between the groups in the rates of fever, injection site reactions or rashes after vaccination. Seroconversion rates and geometric mean titers for measles, mumps and rubella were not significantly different between groups. The varicella seroconversion rate and percentage with glycoprotein-based ELISA titers > or = 5.0 units were similar between the two groups (99.5 and 92.5% vs. 100 and 94.8% for Groups A and B, respectively), but the geometric mean titers were statistically significantly different (13.2 for Group A and 17.9 for Group B). Varicella antibody persistence rates were >98 to 100% during 6 years of follow-up in both groups. Compared with historical rates, varicella vaccine efficacy during 5 years was estimated to be 90.5% (95% confidence interval, 86.2%, 95.0%) and 88.9% (95% confidence interval, 83.7%, 93.7%) in Groups A and B, respectively. CONCLUSIONS: Administration of M-M-R II and VARIVAX concomitantly at separate injection sites or 6 weeks apart was generally well-tolerated and immunogenic in healthy children 12 months to 6 years of age. VARIVAX administered with M-M-R II induced persistent immunity and long-term protection against breakthrough varicella infection.

Naturally occurring triggers that induce apoptosis-like programmed cell death in Plasmodium berghei ookinetes.

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy.

In vivo evaluation of a biolimus eluting nickel titanium self expanding stent with overlapping balloon expandable drug eluting and bare metal stents in a porcine coronary model.

AIMS: Long lesions and complex vessel anatomy frequently require the use of overlapping stents to treat a lesion. The purpose of this study was to evaluate the long-term effects of overlapping the Axxess Biolimus A9 eluting stent (BES) with two of the most commonly used, commercially available drug eluting stents. These stents were compared to BxVelocity bare metal (BMS) stents in a porcine coronary stent-injury model. METHODS AND RESULTS: Nineteen juvenile farm swine, 25-35 kg in weight, 3-6 months in age were utilised. Each animal received an Axxess stent to their coronary artery as permitted by the individual animal's anatomy. A second stent, either a Cypher, sirolimus eluting stent (SES) or, a Taxus, paclitaxel eluting stent (PES), or a BxVelocity bare metal stent (BMS) were implanted in an overlapped fashion. The animals were then followed for either 28 or 180 days as specified by a randomisation scheme. At the end of each follow-up period, they were euthenised, and the vessels containing the overlapping stents were harvested, processed into histological sections, and analysed. Compared to bare metal stents, overlapped segments using DES exhibited delayed vascular healing compared to both the proximal and distal non-overlap sites at each of the follow-up time point. Overall, in the non-overlap stent segments, SES induced significantly more inflammation and neointimal hyperplasia compared to PES and BMS. CONCLUSIONS: In this study of BMS and two different types of DES overlapped with the Axxess Biolimus A9 eluting stent, we found that while there was a delay in the degree of vascular healing with DES compared to BMS, the specific type of DES that was overlapped with BES did not affect the behaviour of the overlap zone in terms of most of the histomorphometric measures at 28 or 180 days. This was true whether the stent was drug eluting or bare metal. More inflammation with delayed healing was seen in the SES compared to PES and BMS.

Long-term effects of novel biolimus eluting DEVAX AXXESS plus nitinol self-expanding stent in a porcine coronary model.

OBJECTIVE: The purpose of this study was to evaluate the long-term effects of the DEVAX AXXESS biolimus eluting stent (BES) in a porcine coronary model, compared with those of bare metal stent (BMS) and polymer only stent (POS) controls. BACKGROUND: Excessive neointimal growth has been identified as a major cause of late failure of percutaneous coronary interventions. The effect of drug eluting from self-expanding stents for prevention of neointimal hyperplasia has not been studied before. The DEVAX AXXESS is a self-expanding nickel titanium stent, coated with antiproliferative compound-biolimus. METHODS: Twenty juvenile farm swine, 25-35 kg in weight, 3-6 months in age were used. Each animal received a stent to the left anterior descending artery, left circumflex or right coronary arteries as permitted per anatomy. The chronic vascular response after BES implantation was compared with that after BMS and POS implantation at 28, 90, and 180 days follow-up. RESULTS: The 28-day outcome by quantitative coronary angiography (QCA) showed significant increase in minimal luminal diameter (MLD) in the BES (MLD: 2.90 +/- 0.97, 2.39 +/- 0.90, 1.59 +/- 0.91; P = 0.009) compared with BMS and POS, respectively. By histomorphometric analysis, there was also a corresponding significant reduction in neointimal tissue proliferation in the BES (average neointimal area: 2.78 +/- 0.07, 5.46 +/- 0.66, 8.42 +/- 0.85; P = 0.002) compared with that in BMS and POS controls, respectively at 28-days follow-up. At 90 and 180 days, the mean neointimal area was not significantly different between the BES and the controls. CONCLUSIONS: BES favorably modulates the neointimal tissue formation for 28 days, in the porcine coronary model. Long-term inhibition of neointimal hyperplasia is not sustained most likely because of the delayed cellular proliferation and inflammation in the vessel wall.