Circulating nerve growth factor levels are increased in humans with allergic diseases and asthma.

Nerve growth factor (NGF) serum levels were measured in 49 patients with asthma and/or rhinoconjunctivitis and/or urticaria-angioedema. Clinical and biochemical parameters, such as bronchial reactivity, total and specific serum IgE levels, and circulating eosinophil cationic protein levels, were evaluated in relation to NGF values in asthma patients. NGF was significantly increased in the 42 allergic (skin-test- or radioallergosorbent-test-positive) subjects (49.7 +/- 28.8 pg/ml) versus the 18 matched controls (3.8 +/- 1.7 pg/ml; P < 0.001). NGF levels in allergic patients with asthma, rhinoconjunctivitis, and urticaria-angioedema were 132.1 +/- 90.8, 17.6 +/- 6.1, and 7.6 +/- 1.8 pg/ml (P < 0.001, P < 0.002, and P < 0.05 versus controls), respectively. Patients with more than one allergic disease had higher NGF serum values than those with a single disease. When asthma patients were considered as a group, NGF serum values (87.6 +/- 59.8 pg/ml) were still significantly higher than those of control groups (P < 0.001), but allergic asthma patients had elevated NGF serum levels compared with nonallergic asthma patients (132.1 +/- 90.8 versus 4.9 +/- 2.9 pg/ml; P < 0.001). NGF serum levels correlate to total IgE serum values (rho = 0.43; P < 0.02). The highest NGF values were found in patients with severe allergic asthma, a high degree of bronchial hyperreactivity, and high total IgE and eosinophil cationic protein serum levels. This study represents the first observation (that we know of) that NGF is increased in human allergic inflammatory diseases and asthma.

Safety and use of sputum induction in children with cystic fibrosis

We assessed the safety and use of induced sputum (IS) in children with cystic fibrosis (CF). Forty-eight children (19 males) with CF, mean age 12.6 (range, 7.3-17.0) years and median forced expired volume in 1 sec (FEV1) 48% (range, 14-77%) predicted were recruited. Patients spontaneously expectorated sputum and then performed sputum induction by inhalation of nebulized 7% hypertonic saline. Samples were sent for bacteriological culture, and for measurement of the following inflammatory mediators: interleukin-8, myeloperoxidase, eosinophil cationic protein, and neutrophil elastase activity. FEV1 was performed before and after inhalation of hypertonic saline. There was no increase in mediator levels in IS compared to expectorated sputum (ES) samples. Only 3 patients demonstrated significant bronchoconstriction following inhalation of hypertonic saline, by the method used. From the ES samples, Pseudomonas aeruginosa was isolated in 13 patients, Staphylococcus aureus in 7 patients, Stenotrophomonas maltophilia in 1 patient, and both Pseudomonas aeruginosa and Staphylococcus aureus in 5 patients. All these organisms were found in the IS samples. However, in 2 patients whose ES grew no organisms, one patient's IS grew Pseudomonas aeruginosa, and the other patient's IS grew Staphylococcus aureus. In our study, sputum induction was safe, with no proinflammatory effect.

Effects of recombinant human DNase and hypertonic saline on airway inflammation in children with cystic fibrosis

Recombinant human DNase (rhDNase) is an established treatment in cystic fibrosis (CF), but it may liberate cationic mediators bound to DNA in the airways. An alternative mucolytic therapy is hypertonic saline (HS); however, HS may potentiate neutrophilic inflammation. We compared the effect of rhDNase and HS on cationic proinflammatory mediators in CF sputum. In a randomized, crossover trial, 48 children with CF were allocated consecutively to 12 weeks of once-daily 2.5 mg rhDNase, alternate-day 2.5 mg rhDNase, and twice-daily 7% HS. Sputum levels of total interleukin-8 (IL-8), free IL-8, myeloperoxidase, eosinophil cationic protein, and neutrophil elastase (NE) activity were measured before and after each treatment. The change in mediator levels from baseline with daily rhDNase and HS was not significant; however, with alternate-day rhDNase, there was an increase in free IL-8. When changes in mediator levels with daily rhDNase were compared with alternate-day rhDNase and HS, no significant differences were detected. Only changes in NE activity were associated with changes in lung function. In summary, we were unable to show that rhDNase or HS promote airway inflammation in CF.

Complementary surface-enhanced resonance raman spectroscopic biodetection of mixed protein solutions by chitosan- and silica-coated plasmon-tuned silver nanoparticles

Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b5, whereas functionalization with SiO2 amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy.

Exhaled nitric oxide correlates with airway eosinophils in childhood asthma

Background: Exhaled nitric oxide has been proposed as a marker for airway inflammation in asthma. The aim of this study was to compare exhaled nitric oxide levels with inflammatory cells and mediators in bronchoalveolar lavage fluid from asthmatic and normal children.

Methods: Children were recruited from elective surgical lists and a non-bronchoscopic bronchoalveolar lavage (BAL) was performed after induction of anaesthesia. Exhaled nitric oxide (parts per billion) was measured by two techniques: tidal breathing and restricted breath.

Results: Median (interquartile range) exhaled nitric oxide measured by restricted breath was increased in asthmatics compared with normal children (24.3 (10.5–66.5) v 9.7 (6.5–16.5), difference between medians 14.6 (95% CI 5.1 to 29.9), p=0.001). In asthmatic children exhaled nitric oxide correlated significantly with percentage eosinophils (r=0.78, p<0.001 (tidal breathing) and r=0.78, p<0.001 (restricted breath)) and with eosinophilic cationic protein (r=0.53, p<0.01 restricted breath)), but not with other inflammatory cells in the BAL fluid. The area under the receiver operator characteristic curves for the prediction of the presence of eosinophilic airways inflammation by exhaled nitric oxide (tidal and restricted) was 0.80 and 0.87, respectively.

Conclusions: Exhaled nitric oxide correlates closely with percentage eosinophils in BAL fluid in asthmatic children and is therefore likely to be a useful non-invasive marker of airway inflammation.

Effect of natural seasonal pollen exposure and repeated nasal allergen provocations on elevation of exhaled nitric oxide

BACKGROUND: Exhaled nitric oxide (FENO) is a marker for allergic airway inflammation. We wondered whether in patients with intermittent allergic rhinitis only (i) natural pollen exposure and (ii) artificial pollen exposure by repeated nasal allergen provocations may lead to an elevation of FENO. METHODS: In two prospective studies, we compared the FENO of nonatopic controls with the FENO of nonasthmatic individuals with mild intermittent rhinitis to tree and/or grass pollen. Study I: 13 atopic individuals and seven controls had measurements of FENO, blood eosinophils and eosinophilic cationic protein (ECP) before, during and after pollen season. Study II: 16 atopic individuals and 12 controls had nasal allergen provocations on four following days out of pollen season, with daily measurements of FENO before, 2 and 6 h after provocation, and determination of blood eosinophils, ECP and FEV1 at baseline, on days 5 and 10-12. RESULTS: Natural pollen exposure (study I) caused a significant elevation of FENO in allergic individuals. Nasal allergen provocations (study II) did not elicit a statistically significant rise neither of FENO nor of blood eosinophils between baseline and day 5. However, a subgroup of four individuals with a rise of blood eosinophils during nasal allergen provocations showed also a rise of FENO. CONCLUSIONS: We suppose that in allergic rhinitis a concomitant reaction of the bronchial system is dependent on a strong local inflammation leading to a generalized immune stimulation.

Estudo das alterações hematológicas e urinárias em cães em diferentes estágios de disfunção renal e avaliação do biomarcador cistatina C

Chapter 2 - Cystatin C is a cationic protein is not glycosylated, produced a steady state for all nucleated and present in biological fluids cells being freely filtered by the glomeruli and almost completely catabolized in the proximal tubule, it is a promising early renal dysfunction marker. This study aimed to determine and compare the serum concentration of cystatin C biomarker in 86 dogs. The animals were divided into four groups according to serum creatinine levels: G1 - up. 1.4 mg / dL (23 animals), G2 - 1.5-2.0 mg / dL (16 animals), G3 - 2.1 to 5.0 mg / dL (24 animals) and G4 - above 5.1 mg / dL (23 animals). There was the measurement of the parameters used in the clinical routine of small animals such as urea, urinary gamma glutamyl transferase, proteinuria, alkaline phosphatase, sodium, potassium, chloride, calcium, phosphorus, calcium/phosphorus ratio and cystatin C. There was no statistical difference for urea, proteinuria, phosphorus, calcium/phosphorus, potassium and cystatin C, however, the other showed no statistical difference. Based on the results we can infer that cystatin C was not a good early indicator of kidney disease in dogs. Chapter 3 - This study aimed to determine the hematological and urinalysis elements such as density, proteinuria, cylinders and pH in 86 dogs The animals were divided into four stages according to serum creatinine levels: I - up to 1.4 mg/dL (23 animals), II - 1.5-2.0 mg/dL (16 animals), III from 2.1 to 5.0 mg/dL (24 animals) and IV - above 5.1 mg/dL (23 animals). In stage III, IV there was anemia normocytic normochromic type. Stage II had a leukocytosis frame by neutrophilia with a regenerative left shift and stage III and IV detour degenerative left. The density remained within the reference values all stages. Proteinuria showed statistical significance for the classification 2+ (1.0 g/L), between stage I and II, II and IV. Only the cylinder granular statistical difference in the classification 2+ between stage II and III, and 3+ between stage I and III. The prevailing pH was acid. The haematological values compared to serum creatinine stages showed the changes in hemoglobin and packed cell volume erythrocytes become more pronounced as serum creatinine values rise , this is also the behavior of neutrophils rods and proteinuria.

Effect of vitamin D supplementation on bone status, glucose homeostasis and immune function in children with vitamin D deficiency

Background: Between 1961-1971 vitamin D deficiency was recognized as a public health issue in the UK, because of the lack of effective sunlight and the population mix [1, 2]. In recent years, health care professionals have cited evidence suggesting a re-emergence of the vitamin D deficiency linked to a number of health consequences as a concern [3-6]. Evidence from observational studies has linked low vitamin D status with impairment in glucose homeostasis and immune dysfunction [7-9]. However, interventional studies, particularly those focused on paediatric populations, have been limited and inconsistent. There is a need for detailed studies, to clarify the therapeutic benefits of vitamin D in these important clinical areas. Objective: The aims of this PhD thesis were two-fold. Firstly, to perform preliminary work assessing the association between vitamin D deficiency and bone status, glucose homeostasis and immune function, and to explore any changes in these parameters following short term vitamin D3 replacement therapy. Secondly, to assess the effectiveness of an electronic surveillance system (ScotPSU) as a tool to determine the current incidence of hospital-based presentation of childhood vitamin D deficiency in Scotland. Methods: Active surveillance was performed for a period of two years as a part of an electronic web-based surveillance programme performed by the Scottish Paediatric Surveillance Unit (ScotPSU). The validity of the system was assessed by identifying cases with profound vitamin D deficiency (in Glasgow and Edinburgh) from the regional laboratory. All clinical details were checked against those identified using the surveillance system. Thirty-seven children aged 3 months to 10 years, who had been diagnosed with vitamin D deficiency, were recruited for the bone, glucose and immunity studies over a period of 24 months. Twenty-five samples were analysed for the glucose and bone studies; of these, 18 samples were further analysed for immune study. Treatment consisted of six weeks taking 5000 IU units cholecalciferol orally once a day. At baseline and after completion of treatment, 25 hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), alkaline phosphatase (ALP), collagen type 1 cross-linked C-telopeptide (CTX), osteocalcin (OCN), calcium, phosphate, insulin, glucose, homeostasis model assessment index, estimated insulin resistance (HOMA IR), glycated hemoglobin (HbA1c), sex hormone binding globulin (SHBG), lipids profiles, T helper 1 (Th1) cytokines (interleukin-2 ( IL-2), tumor necrosis factors-alpha (TNF-α), interferon-gamma (INF-γ)), T helper 2 (Th2) cytokines (interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6)), T helper 17 (Th17) cytokine (interleukin-17 (IL-17)), Regulatory T (Treg) cytokine (interleukin-10 (IL-10)) and chemokines/cytokines, linked with Th1/Th2 subset balance and/or differentiation (interleukin-8 (IL-8), interleukin-12 (IL-12), eosinophil chemotactic protein ( EOTAXIN), macrophage inflammatory proteins-1beta (MIP-1β), interferon-gamma-induced protein-10 (IP-10), regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1(MCP-1)) were measured. Leukoocyte subset analysis was performed for T cells, B cells and T regulatory cells and a luminex assay was used to measure the cytokiens. Results: Between September 2009 and August 2011, 163 cases of vitamin D deficiency were brought to the attention of the ScotPSU, and the majority of cases (n = 82) were reported in Glasgow. The cross-validation checking in Glasgow and Edinburgh over a one-year period revealed only 3 (11%) cases of clearly symptomatic vitamin D deficiency, which had been missed by the ScotPSU survey in Glasgow. While 16 (67%) symptomatic cases had failed to be reported through the ScotPSU survey in Edinburgh. For the 23 children who are included in bone and glucose studies, 22 (96%) children had basal serum 25(OH)D in the deficiency range (< 50 nmol/l) and one (4%) child had serum 25(OH)D in the insufficiency range (51-75 nmol/l). Following vitamin D3 treatment, 2 (9%) children had final serum 25(OH)D lower than 50 nmol/l, 6 (26%) children had final serum 25(OH)D between >50-75 nmol/l, 12 (52%) children reached a final serum 25(OH)D >75-150 nmol/l and finally 3 (13%) exceeded the normal reference range with a final 25(OH)D >150 nmol/l. Markers for remodelling ALP and PTH had significantly decreased (p = 0.001 and <0.0001 for ALP and PTH respectively). In 17 patients for whom insulin and HOMA IR data were available and enrolled in glucose study, significant improvements in insulin resistance (p = 0.04) with a trend toward a reduction in serum insulin (p = 0.05) was observed. Of those 14 children who had their cytokines profile data analysed and enrolled in the immunity study, insulin and HOMA IR data were missed in one child. A significant increase in the main Th2 secreted cytokine IL-4 (p = 0.001) and a tendency for significant increases in other Th2 secreted cytokines IL-5 (p = 0.05) and IL-6 (p = 0.05) was observed following vitamin D3 supplementation. Conclusion: An electronic surveillance system can provide data for studying the epidemiology of vitamin D deficiency. However, it may underestimate the number of positive cases. Improving vitamin D status in vitamin D deficient otherwise healthy children significantly improved their vitamin D deficient status, and was associated with an improvement in bone profile, improvements in insulin resistance and an alteration in main Th2 secreting cytokines.

Protein Assembly onto Cationic Supported Bilayers

Cationic supported bilayers on latex are useful to isolate and immobilize oppositely charged proteins as a monomolecular layer over a range of low protein concentrations and particle number densities. Cholera toxin (CT) from Vibrio cholerae, an 87 kDa AB(5) hexameric protein and bovine serum albumin (BSA) self-assembled on dioctadecyldimethylammonium bromide (DODAB) supported bilayers with high affinity yielding highly organized and monodisperse particulates at 5 x 10(9) particles/mL, over a range of low protein concentrations (0-0.025 mg/mL BSA or CT). Protein association onto the bilayer-covered polystyrene sulfate (PSS) was determined from adsorption isotherms, dynamic light scattering for size distributions and zeta-potential analysis revealing a monomolecular, thin and highly organized protein layer surrounding each particle with potential for biospecific recognition such as antigen-antibody, receptor-ligand, hybridization of oligonucleotide sequences, all of them important in immunodiagnosis, selective biomolecular chromatographic separations, microarrays design and others.

Small cationic DDA:TDB liposomes as protein vaccine adjuvants obviate the need for TLR agonists in inducing cellular and humoral responses

Most subunit vaccines require adjuvants in order to induce protective immune responses to the targeted pathogen. However, many of the potent immunogenic adjuvants display unacceptable local or systemic reactogenicity. Liposomes are spherical vesicles consisting of single (unilamellar) or multiple (multilamellar) phospholipid bi-layers. The lipid membranes are interleaved with an aqueous buffer, which can be utilised to deliver hydrophilic vaccine components, such as protein antigens or ligands for immune receptors. Liposomes, in particular cationic DDA:TDB vesicles, have been shown in animal models to induce strong humoral responses to the associated antigen without increased reactogenicity, and are currently being tested in Phase I human clinical trials. We explored several modifications of DDA:TDB liposomes--including size, antigen association and addition of TLR agonists--to assess their immunogenic capacity as vaccine adjuvants, using Ovalbumin (OVA) protein as a model protein vaccine. Following triple homologous immunisation, small unilamellar vesicles (SUVs) with no TLR agonists showed a significantly higher capacity for inducing spleen CD8 IFN? responses against OVA in comparison with the larger multilamellar vesicles (MLVs). Antigen-specific antibody reponses were also higher with SUVs. Addition of the TLR3 and TLR9 agonists significantly increased the adjuvanting capacity of MLVs and OVA-encapsulating dehydration-rehydration vesicles (DRVs), but not of SUVs. Our findings lend further support to the use of liposomes as protein vaccine adjuvants. Importantly, the ability of DDA:TDB SUVs to induce potent CD8 T cell responses without the need for adding immunostimulators would avoid the potential safety risks associated with the clinical use of TLR agonists in vaccines adjuvanted with liposomes.

Cationic liposomes as adjuvants for subunit protein vaccines:their role in the depot effect and antigen processing by APCs

Introduction: The requirement of adjuvants in subunit protein vaccination is well known yet their mechanisms of action remain elusive. Of the numerous mechanisms suggested, cationic liposomes appear to fulfil at least three: the antigen depot effect, the delivery of antigen to antigen presenting cells (APCs) and finally the danger signal. We have investigated the role of antigen depot effect with the use of dual radiolabelling whereby adjuvant and antigen presence in tissues can be quantified. In our studies a range of cationic liposomes and different antigens were studied to determine the importance of physical properties such as liposome surface charge, antigen association and inherent lipid immunogenicity. More recently we have investigated the role of liposome size with the cationic liposome formulation DDA:TDB, composed of the cationic lipid dimethyldioctadecylammonium (DDA) and the synthetic mycobacterial glycolipid trehalose 6,6’-dibehenate (TDB). Vesicle size is a frequently investigated parameter which is known to result in different routes of endocytosis. It has been postulated that targeting different routes leads to different intracellular signaling pathway activation and it is certainly true that numerous studies have shown vesicle size to have an effect on the resulting immune responses (e.g. Th1 vs. Th2). Aim: To determine the effect of cationic liposome size on the biodistribution of adjuvant and antigen, the ensuing humoral and cell-mediated immune responses and the uptake and activation of antigen by APCs including macrophages and dendritic cells. Methods: DDA:TDB liposomes were made to three different sizes (~ 0.2, 0.5 and 2 µm) followed by the addition of tuberculosis antigen Ag85B-ESAT-6 therefore resulting in surface adsorption. Liposome formulations were injected into Balb/c or C57Bl/6 mice via the intramuscular route. The biodistribution of the liposome formulations was followed using dual radiolabelling. Tissues including muscle from the site of injection and local draining lymph nodes were removed and liposome and antigen presence quantified. Mice were also immunized with the different vaccine formulations and cytokine production (from Ag85B-ESAT-6 restimulated splenocytes) and antibody presence in blood assayed. Furthermore, splenocyte proliferation after restimulating with Ag85B-ESAT-6 was measured. Finally, APCs were compared for their ability to endocytose vaccine formulations and the effect this had on the maturation status of the cell populations was compared. Flow cytometry and fluorescence labelling was used to investigate maturation marker up-regulation and efficacy of phagocytosis. Results: Our results show that for an efficient Ag85B-ESAT-6 antigen depot at the injection site, liposomes composed of DDA and TDB are required. There is no significant change in the presence of liposome or antigen at 6hrs or 24hrs p.i, nor does liposome size have an effect. Approximately 0.05% of the injected liposome dose is detected in the local draining lymph node 24hrs p.i however protein presence is low (<0.005% dose). Preliminary in vitro data shows liposome and antigen endocytosis by macrophages; further studies on this will be presented in addition to the results of the immunisation study.

Formulation and development of cationic liposomes as adjuvants for subunit protein vaccinese

Liposomes remain at the forefront of vaccine design due to their well documented abilities to act as delivery vehicles and adjuvants. Liposomes have been described to initiate an antigen depot-effect, thereby increasing antigen exposure to circulating antigen-presenting cells. More recently, in-depth reviews have focussed on inherent immunostimulatory abilities of various cationic lipids, the use of which is consequently of interest in the development of subunit protein vaccines which when delivered without an adjuvant are poorly immunogenic. The importance of liposomes for the mediation of an antigen depot-effect was examined by use of a dual-radiolabelling technique thereby allowing simultaneous detection of liposomal and antigenic components and analysis of their pharmacokinetic profile. In addition to investigating the biodistribution of these formulations, their physicochemical properties were analysed and the ability of the various liposome formulations to elicit humoral and cell-mediated immune responses was investigated. Our results show a requirement of cationic charge and medium/strong levels of antigen adsorption to the cationic liposome in order for both a liposome and antigen depot-effect to occur at the injection site. The choice of injection route had little effect on the pharmacokinetics or immunogenicity observed. In vitro, cationic liposomes were more cytotoxic than neutral liposomes due to significantly enhanced levels of cell uptake. With regards to the role of bilayer fluidity, liposomes expressing more rigid bilayers displayed increased retention at the injection site although this did not necessarily result in increased antigen retention. Furthermore, liposome bilayer rigidity did not necessarily correlate with improved immunogenicity. In similar findings, liposome size did not appear to control liposome or antigen retention at the injection site. However, a strong liposome size correlation between splenocyte proliferation and production of IL-10 was noted; specifically immunisation with large liposomes lead to increased levels of splenocyte proliferation coupled with decreased IL-10 production.

A comparative study of cationic liposome and niosome-based adjuvant systems for protein subunit vaccines:characterisation, environmental scanning electron microscopy and immunisation studies in mice

Vesicular adjuvant systems composing dimethyldioctadecylammonium (DDA) can promote both cell-mediated and humoral immune responses to the tuberculosis vaccine fusion protein in mice. However, these DDA preparations were found to be physically unstable, forming aggregates under ambient storage conditions. Therefore there is a need to improve the stability of such systems without undermining their potent adjuvanticity. To this end, the effect of incorporating non-ionic surfactants, such as 1-monopalmitoyl glycerol (MP), in addition to cholesterol (Chol) and trehalose 6,6′-dibehenate (TDB), on the stability and efficacy of these vaccine delivery systems was investigated. Differential scanning calorimetry revealed a reduction in the phase transition temperature (T c) of DDA-based vesicles by ∼12°C when MP and cholesterol (1:1 molar ratio) were incorporated into the DDA system. Transmission electron microscopy (TEM) revealed the addition of MP to DDA vesicles resulted in the formation of multi-lamellar vesicles. Environmental scanning electron microscopy (ESEM) of MP-Chol-DDA-TDB (16:16:4:0.5 μmol) indicated that incorporation of antigen led to increased stability of the vesicles, perhaps as a result of the antigen embedding within the vesicle bilayers. At 4°C DDA liposomes showed significant vesicle aggregation after 28 days, although addition of MP-Chol or TDB was shown to inhibit this instability. Alternatively, at 25°C only the MP-based systems retained their original size. The presence of MP within the vesicle formulation was also shown to promote a sustained release of antigen in-vitro. The adjuvant activity of various systems was tested in mice against three subunit antigens, including mycobacterial fusion protein Ag85b-ESAT-6, and two malarial antigens (Merozoite surface protein 1, MSP1, and the glutamate rich protein, GLURP). The MP- and DDA-based systems induced antibody responses at comparable levels whereas the DDA-based systems induced more powerful cell-mediated immune responses. © 2006 The Authors.

Microparticle-mediated gene delivery for the enhanced expression of a 19-KDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP119) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP119 is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h1. High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 lm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA-mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.

Cationic Amino Acid Transporters and Salmonella Typhimurium ArgT Collectively Regulate Arginine Availability towards Intracellular Salmonella Growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.