Intracellular Targeting Specificity of Novel Phthalocyanines Assessed in a Host-Parasite Model for Developing Potential Photodynamic Medicine

Photodynamic therapy, unlikely to elicit drug-resistance, deserves attention as a strategy to counter this outstanding problem common to the chemotherapy of all diseases. Previously, we have broadened the applicability of this modality to photodynamic vaccination by exploiting the unusual properties of the trypanosomatid protozoa, Leishmania, i.e., their innate ability of homing to the phagolysosomes of the antigen-presenting cells and their selective photolysis therein, using transgenic mutants endogenously inducible for porphyrin accumulation. Here, we extended the utility of this host-parasite model for in vitro photodynamic therapy and vaccination by exploring exogenously supplied photosensitizers. Seventeen novel phthalocyanines (Pcs) were screened in vitro for their photolytic activity against cultured Leishmania. Pcs rendered cationic and soluble (csPcs) for cellular uptake were phototoxic to both parasite and host cells, i.e., macrophages and dendritic cells. The csPcs that targeted to mitochondria were more photolytic than those restricted to the endocytic compartments. Treatment of infected cells with endocytic csPcs resulted in their accumulation in Leishmania-containing phagolysosomes, indicative of reaching their target for photodynamic therapy, although their parasite versus host specificity is limited to a narrow range of csPc concentrations. In contrast, Leishmania pre-loaded with csPc were selectively photolyzed intracellularly, leaving host cells viable. Pre-illumination of such csPc-loaded Leishmania did not hinder their infectivity, but ensured their intracellular lysis. Ovalbumin (OVA) so delivered by photo-inactivated OVA transfectants to mouse macrophages and dendritic cells were co-presented with MHC Class I molecules by these antigen presenting cells to activate OVA epitope-specific CD8+T cells. The in vitro evidence presented here demonstrates for the first time not only the potential of endocytic csPcs for effective photodynamic therapy against Leishmania but also their utility in photo-inactivation of Leishmania to produce a safe carrier to express and deliver a defined antigen with enhanced cell-mediated immunity

IgG antibody responses to Plasmodium falciparum merozoite antigens in Kenyan children have a short half-life

BACKGROUND: Data suggest that antibody responses to malaria parasites merozoite antigens are generally short-lived and this has implications for serological studies and malaria vaccine designs. However, precise data on the kinetics of these responses is lacking. METHODS: IgG1 and IgG3 responses to five recombinant Plasmodium falciparum merozoite antigens (MSP-119, MSP-2 type A and B, AMA-1 ectodomain and EBA-175 region II) among Kenyan children were monitored using ELISA for 12 weeks after an acute episode of malaria and their half-lives estimated using an exponential decay model. RESULTS: The responses peaked mainly at week 1 and then decayed rapidly to very low levels within 6 weeks. Estimation of the half-lives of 40 IgG1 responses yielded a mean half-life of 9.8 days (95% CI: 7.6-12.0) while for 16 IgG3 responses it was 6.1 days (95% CI: 3.7-8.4), periods that are shorter than those normally described for the catabolic half-life of these antibody subclasses. CONCLUSION: This study indicates antibodies against merozoite antigens have very short half-lives and this has to be taken into account when designing serological studies and vaccines based on the antigens

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λmax) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages

Artemisinin-naphthoquine combination (ARCO™) therapy for uncomplicated falciparum malaria in adults of Papua New Guinea: A preliminary report on safety and efficacy

<p>Abstract</p> <p>Background</p> <p>The use of anti-malarial drug combinations with artemisinin or with one of its derivatives is now widely recommended to overcome drug resistance in falciparum as well as vivax malaria. The fixed oral dose artemisinin-naphthoquine combination (ANQ, ARCO™) is a newer artemisinin-based combination (ACT) therapy undergoing clinical assessment. A study was undertaken to assess the safety, efficacy and tolerability of ANQ combination in areas of multi-drug resistance to generate preliminary baseline data in adult population of Papua New Guinea.</p> <p>Methods</p> <p>The clinical assessment was an open-labeled, two-arm, randomized study comparing ANQ combination as a single dose regimen and three days regimen (10 mg/kg/day) of chloroquine plus single dose sulphadoxine-pyrimethamine (CQ+SP) for the treatment of uncomplicated falciparum malaria with 28 days follow-up in an adult population. The primary outcome measures for efficacy were day 1, 2, 3 7, 14 and 28-day cure rates. Secondary outcomes included parasite clearance time, fever clearance time, and gametocyte carriage. The main outcome measures for safety were incidences of post-treatment clinical and laboratory adverse events.</p> <p>Results</p> <p>Between June 2005 and July 2006, 130 patients with confirmed uncomplicated <it>P. falciparum </it>were randomly assigned to receive ANQ and CQ+SP, only 100 patients (51 in ANQ group and 49 in CQ+SP group) were evaluated for clinical and parasitological outcomes. All the patients treated with ANQ and CQ+SP showed adequate clinical and parasitological response with 28 days follow-up. The cure rate for ANQ on day 1, 2, 3, 7, 14, and 28 was 47%, 86%, 92%, 94%, 94% and 94%, respectively. Recrudescence account for 6%; all were cleared on day 21. For CQ+SP treated group the cure rates were 24%, 67%, 82%, 82%, 84% and 88%, respectively. Recrudescence accounted for 10%; all were cleared on day 28 except for one patient. Both regimens were well tolerated with no serious adverse events. The proportion of gametocyte carriers was higher in CQ+SP treated group than ANQ treatment (41% versus 12%; p < 0.05).</p> <p>Conclusion</p> <p>While these data are not themselves sufficient, it strongly suggests that the ANQ combination as a single dose administration is safe and effective for the treatment of uncomplicated <it>P. falciparum </it>malaria in the adult population of Papua New Guinea and deserves further clinical evaluation.</p

Antigen-Specific B Memory Cell Responses to Plasmodium falciparum Malaria Antigens and Schistosoma haematobium Antigens in Co-Infected Malian Children

Polyparasitism is common in the developing world. We have previously demonstrated that schistosomiasis-positive (SP) Malian children have age-dependent protection from malaria compared to matched schistosomiasis-negative (SN) children. Evidence of durable immunologic memory to malaria antigens is conflicting, particularly in young children and the effect of concomitant schistomiasis upon acquisition of memory is unknown. We examined antigen-specific B memory cell (MBC) frequencies (expressed as percentage of total number of IgG-secreting cells) in 84 Malian children aged 4–14 to malaria blood-stage antigens, apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP-1) and to schistosomal antigens, Soluble Worm Antigenic Preparation (SWAP) and Schistosoma Egg Antigen (SEA), at a time point during the malaria transmission season and a follow-up dry season visit. We demonstrate, for the first time, MBC responses to S. haematobium antigens in Malian children with urinary egg excretion and provide evidence of seasonal acquisition of immunologic memory, age-associated differences in MBC acquisition, and correlation with circulating S. haematobium antibody. Moreover, the presence of a parasitic co-infection resulted in older children, aged 9–14 years, with underlying S. haematobium infection having significantly more MBC response to malaria antigens (AMA1 and MSP1) than their age-matched SN counterparts. We conclude that detectable MBC response can be measured against both malaria and schistosomal antigens and that the presence of S. haematobium may be associated with enhanced MBC induction in an age-specific manner

2D-DIGE as a strategy to identify serum biomarkers in Mexican patients with Type-2 diabetes with different body mass index

"Obesity and type 2 diabetes (T2D) are the most prevalent and serious metabolic diseases affecting people worldwide. However racial and ethnic disparities seems to be a risk factor for their development. Mexico has been named as one of the largest populations with the highest prevalence of diabetes and obesity. The aim of this study was to identify novel T2D-associated proteins in Mexican patients. Blood samples were collected from 62 Mexican patients with T2D and they were grouped according to their body mass index (BMI). A panel of 10 diabetes and obesity serum markers was determined using MAGPIX. A comparative proteomics study was performed using two-dimensional difference in-gel electrophoresis (2D-DIGE) followed by mass spectrometry (LC-MS/MS). We detected 113 spots differentially accumulated, in which 64 unique proteins were identified, proteins that were involved in metabolism pathways, molecular transport, and cellular signalling. Four proteins (14-3-3, ApoH, ZAG, and OTO3) showing diabetes-related variation and also changes in relation to obesity were selected for further validation by western blotting. Our results reveal new diabetes related proteins present in the Mexican population. These could provide additional insight into the understanding of diabetes development in Mexican population and may also be useful candidate biomarkers.

Daily egg consumption in hyperlipidemic adults - Effects on endothelial function and cardiovascular risk

<p>Abstract</p> <p>Background</p> <p>Limiting consumption of eggs, which are high in cholesterol, is generally recommended to reduce risk of cardiovascular disease. However, recent evidence suggests that dietary cholesterol has limited influence on serum cholesterol or cardiac risk.</p> <p>Objective</p> <p>To assess the effects of egg consumption on endothelial function and serum lipids in hyperlipidemic adults.</p> <p>Methods</p> <p>Randomized, placebo-controlled crossover trial of 40 hyperlipidemic adults (24 women, 16 men; average age = 59.9 ± 9.6 years; weight = 76.3 ± 21.8 kilograms; total cholesterol = 244 ± 24 mg/dL). In the acute phase, participants were randomly assigned to one of the two sequences of a single dose of three medium hardboiled eggs and a sausage/cheese breakfast sandwich. In the sustained phase, participants were then randomly assigned to one of the two sequences of two medium hardboiled eggs and 1/2 cup of egg substitute daily for six weeks. Each treatment assignment was separated by a four-week washout period. Outcome measures of interest were endothelial function measured as flow mediated dilatation (FMD) and lipid panel.</p> <p>Results</p> <p>Single dose egg consumption had no effects on endothelial function as compared to sausage/cheese (0.4 ± 1.9 vs. 0.4 ± 2.4%; <it>p </it>= 0.99). Daily consumption of egg substitute for 6 weeks significantly improved endothelial function as compared to egg (1.0 ± 1.2% vs. -0.1 ± 1.5%; <it>p </it>< 0.01) and lowered serum total cholesterol (-18 ± 18 vs. -5 ± 21 mg/dL; <it>p </it>< 0.01) and LDL (-14 ± 20 vs. -2 ± 19 mg/dL; <it>p </it>= 0.01). Study results (positive or negative) are expressed in terms of change relative to baseline.</p> <p>Conclusions</p> <p>Egg consumption was found to be non-detrimental to endothelial function and serum lipids in hyperlipidemic adults, while egg substitute consumption was beneficial.</p

Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

Background The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. Methodology/Principal Findings We report the 1.8 A crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Conclusions/Significance Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.National Health and Medical Research Council (Australia) (NHMRC grant 488502)National Institutes of Health (U.S.) (Grant GM62414-0 )Ontario. Ministry of Revenue (Challenge Fund