Differences in vector-genome processing and illegitimate integration of non-integrating lentiviral vectors.

A variety of mutations in lentiviral vector expression systems have been shown to generate a non-integrating phenotype. We studied a novel 12 base-pair U3-long terminal repeats (LTR) integrase (IN) attachment site deletion (U3-LTR att site) mutant and found similar physical titers to the previously reported IN catalytic core mutant IN/D116N. Both mutations led to a greater than two log reduction in vector integration; with IN/D116N providing lower illegitimate integration frequency, whereas the U3-LTR att site mutant provided a higher level of transgene expression. The improved expression of the U3-LTR att site mutant could not be explained solely based on an observed modest increase in integration frequency. In evaluating processing, we noted significant differences in unintegrated vector forms, with the U3-LTR att site mutant leading to a predominance of 1-LTR circles. The mutations also differed in the manner of illegitimate integration. The U3-LTR att site mutant vector demonstrated IN-mediated integration at the intact U5-LTR att site and non-IN-mediated integration at the mutated U3-LTR att site. Finally, we combined a variety of mutations and modifications and assessed transgene expression and integration frequency to show that combining modifications can improve the potential clinical utility of non-integrating lentiviral vectors.

Low serum 25-hydroxyvitamin D is associated with increased risk of stress fracture during Royal Marine recruit training.

UNLABELLED: The aim of this study was to investigate vitamin D status and stress fracture risk during Royal Marine military training. Poor vitamin D status was associated with an increased risk of stress fracture. Vitamin D supplementation may help to reduce stress fracture risk in male military recruits with low vitamin D status. INTRODUCTION: Stress fracture is a common overuse injury in military recruits, including Royal Marine (RM) training in the UK. RM training is recognised as one of the most arduous basic training programmes in the world. Associations have been reported between serum 25-hydroxyvitamin D (25(OH)D) and risk of stress fracture, but the threshold of 25(OH)D for this effect remains unclear. We aimed to determine if serum 25(OH)D concentrations were associated with stress fracture risk during RM training. METHODS: We prospectively followed 1082 RM recruits (males aged 16-32 years) through the 32-week RM training programme. Troops started training between September and July. Height, body weight and aerobic fitness were assessed at week 1. Venous blood samples were drawn at weeks 1, 15 and 32. Serum samples were analysed for 25(OH)D and parathyroid hormone (PTH). RESULTS: Seventy-eight recruits (7.2 %) suffered a total of 92 stress fractures. Recruits with a baseline serum 25(OH)D concentration below 50 nmol L(-1) had a higher incidence of stress fracture than recruits with 25(OH)D concentration above this threshold (χ(2) (1) = 3.564, p = 0.042; odds ratio 1.6 (95 % confidence interval (CI) 1.0-2.6)). Baseline serum 25(OH)D varied from 47.0 ± 23.7 nmol L(-1) in February, to 97.3 ± 24.6 nmol L(-1) in July (overall mean 69.2 ± 29.2 nmol L(-1), n = 1016). There were weak inverse correlations between serum 25(OH)D and PTH concentrations at week 15 (r = -0.209, p < 0.001) and week 32 (r = -0.214, p < 0.001), but not at baseline. CONCLUSION: Baseline serum 25(OH)D concentration below 50 nmol L(-1) was associated with an increased risk of stress fracture. Further studies into the effects of vitamin D supplementation on stress fracture risk are certainly warranted.

Staphylococcus aureus colonization and acquisition of skin and soft tissue infection among Royal Marines recruits: a prospective cohort study.

OBJECTIVES: Skin and soft tissue infections (SSTIs) are a serious health issue for military personnel. Of particular importance are those caused by methicillin-resistant Staphylococcus aureus and Panton-Valentine leucocidin (PVL)-positive S. aureus (PVL-SA), as they have been associated with outbreaks of SSTIs. A prospective observational study was conducted in Royal Marine (RM) recruits to investigate the prevalence of PVL-SA carriage and any association with SSTIs. METHODS: A total of 1012 RM recruits were followed through a 32-week training programme, with nose and throat swabs obtained at weeks 1, 6, 15 and 32. S. aureus isolates were characterized by antibiotic susceptibility testing, spa typing, presence of mecA/C and PVL genes. Retrospective review of the clinical notes for SSTI acquisition was conducted. RESULTS: S. aureus colonization decreased from Week 1 to Week 32 (41% to 26%, p < 0.0001). Of 1168 S. aureus isolates, three out of 1168 (0.3%) were MRSA and ten out of 1168 (0.9%) PVL-positive (all MSSA) and 169 out of 1168 (14.5%) were resistant to clindamycin. Isolates showed genetic diversity with 238 different spa types associated with 25 multi-locus sequence type (MLST) clonal complexes. SSTIs were seen in 35% (351/989) of recruits with 3 training days lost per recruit. SSTI acquisition rate was reduced amongst persistent carriers (p < 0.0283). CONCLUSIONS: Nose and throat carriage of MRSA and PVL-SA was low among recruits, despite a high incidence of SSTIs being reported, particularly cellulitis. Carriage strains were predominantly MSSA with a marked diversity of genotypes. Persistent nose and/or throat carriage was not associated with SSTI acquisition. Putative person-to-person transmission within troops was identified based on spa typing requiring further research to confirm and explore potential transmission routes.

Mechanisms of U46619- and 5-HT-induced contraction of bovine pulmonary arteries: role of chloride ions.

BACKGROUND AND PURPOSE: Thromboxane A(2) and 5-hydroxytryptamine (5-HT) are implicated in pulmonary hypertension. The involvement of chloride, voltage-operated calcium channels (VOCCs), store-operated calcium channels (SOCCs) and the Rho kinase in the contractile response of bovine pulmonary arteries (BPA) to the thromboxane A(2) mimetic U46619 and 5-HT was investigated. EXPERIMENTAL APPROACH: Endothelium-intact ring segments of BPA were mounted in Krebs/Henseleit buffer (37 degrees C) under a tension of 2g and gassed with 95%O(2)/5%CO(2). KEY RESULTS: Depletion or removal of extracellular chloride, inhibition of chloride and SOCC, Na:K:2Cl, Cl/HCO(3), Rho kinase inhibited contractions to U46619. Combining Rho kinase inhibition and chloride channel blockade (with NPPB) almost abolished the contractions to U46619. In contrast 5-HT-induced contraction was inhibited by verapamil and mibefradil. Depletion of stored calcium with caffeine almost abolished the response to U46619 but not 5-HT. The contraction by the sarco(endo)plasmic reticulum Ca(2+)-ATPase inhibitor CPA was abolished by SOCC and chloride channel blockade (with NPPB) and by chloride depletion. CONCLUSIONS AND IMPLICATIONS: This study suggests that the contractile response of BPA to U46619 involves Rho kinase together with a chloride-sensitive mechanism, which does not involve VOCC but may have a role in calcium release and calcium entry via SOCC. In contrast contraction of the BPA by 5-HT appears to involve verapamil- and mibefradil-sensitive VOCC. This study may indicate that the use of calcium channel blockers in the management of pulmonary hypertension may not always be effective and that Rho kinase and chloride channels may be targets for the development of new therapies.

Predicting response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer with serum biomarkers.

Introduction The aim of this study was to identify patient factors including serum biomarkers that may predict response to neoadjuvant chemoradiotherapy (CRT) in patients with locally advanced rectal cancer staged on magnetic resonance imaging. Prediction of response may be helpful when selecting patients for a non-operative programme. Methods A retrospective review was carried out of patients undergoing neoadjuvant CRT for rectal cancer, conducted at the Royal Devon and Exeter Hospital. All patients were managed through the multidisciplinary team. Receiver operating characteristic (ROC) curve analysis was undertaken to assess the ability of biomarkers to predict response to neoadjuvant CRT. The biomarkers assessed included neutrophils, lymphocytes, monocytes, haemoglobin, platelets, C-reactive protein and carcinoembryonic antigen. Results Seventy-three patients underwent neoadjuvant CRT between January 2006 and December 2011. Nine (12.3%) of these experienced a clinical complete response and were managed with a 'watch and wait' approach. An additional ten patients (13.7%) had a pathological complete response following surgery. Using ROC curve analysis, the biomarkers with the largest area under the curve (AUC) were pre-CRT haemoglobin and post-CRT lymphocyte concentrations, producing AUC values of 0.673 and 0.618 respectively for clinical complete response. Pre-CRT haemoglobin and neutrophil concentrations produced the highest AUC values for pathological complete response at 0.591 and 0.614 respectively. Conclusions None of the assessed biomarkers offer the ability to predict response to neoadjuvant CRT in patients with rectal cancer. They cannot therefore assist in identifying complete clinical or pathological responders who could be considered for a non-operative, observational approach.

Body dysmorphic disorder symptoms and risk for suicide: The role of depression.

Body dysmorphic disorder (BDD) is associated with elevated suicidality. Little is known about why BDD patients are at increased risk. The interpersonal-psychological theory of suicide (IPTS) could clarify suicidality in BDD, and theorizes that perceived burdensomeness and thwarted belongingness lead to suicidal desire, while an acquired capability for suicide is necessary to attempt suicide. No study has investigated how BDD symptoms relate to IPTS constructs or mediators of the relationship between BDD and suicidality. Individuals (N=235) enrolled in Amazon.com's Mechanical Turk (MTurk), who had appearance concerns, completed questionnaires about BDD, depression, eating pathology, and suicide risk. MTurk is an online data collection platform in which participants complete surveys for payment. BDD symptoms predicted suicidal desire, but not acquired capability for suicide. Depression mediated the relationship between BDD and suicidal desire. Research should examine how fluctuations in BDD affect suicide risk. Replication in a clinical sample may inform treatments for BDD.

Sodium-proton exchange across the apical membrane of the alveolar type II cell of the fetal sheep.

In order to detect and characterise Na(+)-H+ countertransport in the fetal lung epithelium we have studied under a variety of conditions the effect of an outward facing H+ gradient on Na+ uptake into purified apical membrane vesicles prepared from alveolar type II cells. Kinetic analysis of the data reveals both a diffusional and a saturable component of total Na+ uptake. Evidence for the presence of a Na(+)-H+ exchanger is demonstrated by (1) stimulation of Na+ uptake by proton loading of vesicles both in the presence and absence of chemical voltage clamping; (2) saturation kinetics with respect to external Na+ with a Km of 16 mM and a Vmax of 2.1 nmol/mg protein per min; (3) amiloride inhibition of Na+ uptake driven by pH gradient. We conclude that although diffusion may be the major component of total Na+ uptake at physiological external Na+ concentration, Na(+)-H+ countertransport provides a possible mechanism for the acidification of fetal lung liquid in-vivo in addition to its established role in intracellular pH and volume regulation.

Hoarding and emotional reactivity: the link between negative emotional reactions and hoarding symptomatology.

Hoarding disorder (HD) is characterized by difficulty discarding, clutter, and frequently excessive acquiring. Theories have pointed to intense negative emotional reactions (e.g., sadness) as one factor that may play a critical role in HD's etiology. Preliminary work with an analogue sample indicated that more intense negative emotions following emotional films were linked with greater hoarding symptoms. Symptom provocation imaging studies with HD patients have also found evidence for excessive activation in brain regions implicated in processing emotions. The current study utilized a sample with self-reported serious hoarding difficulties to examine how hoarding symptoms related to both general and hoarding-related emotional reactivity, taking into account the specificity of these relationships. We also examined how two cognitive factors, fear of decision-making and confidence in memory, modified this relationship. 628 participants with self-identified hoarding difficulties completed questionnaires about general emotional reactivity, depression, anxiety, decision-making, and confidence in memory. To assess hoarding-related emotional reactivity, participants reported their emotional reactions when imagining discarding various items. Heightened general emotional reactivity and more intense emotional reactions to imagined discarding were associated with both difficulty discarding and acquisition, but not clutter, controlling for age, gender, and co-occurring mood and anxiety symptoms. Fear of decision-making and confidence in memory interacted with general emotional reactivity to predict hoarding symptoms. These findings provide support for cognitive-behavioral models of hoarding. Experimental research should be conducted to discover whether emotional reactivity increases vulnerability for HD. Future work should also examine whether emotional reactivity should be targeted in interventions for hoarding.

Regulation of platelet cytosolic free calcium by cyclic nucleotides and protein kinase C.

PAF elicits a rapid, concentration-dependent elevation of platelet cytosolic free calcium ([Caf]), measured by quin2. Elevation of [Caf] is transient, and the rate of reversal increases with agonist concentration. Adenylate cyclase stimulants (PGI2, PGD2) and 8-bromo cAMP; a guanylate cyclase stimulant (sodium nitroprusside) and 8-bromo cGMP; and a protein kinase C stimulant (phorbol myristate acetate) block the elevation of [Caf] induced by PAF, and accelerate its reversal. These results suggest that cAMP, cGMP and 1,2-diacylglycerol (DAG) could act as second messengers to regulate [Caf] in platelets. As PAF is known to stimulate platelet phosphoinositide hydrolysis (ergo DAG formation) but fails to elevate platelet cAMP or cGMP, it is proposed that DAG, via activation of protein kinase C, may act as an endogenous modulator of platelet [Caf]: an action that contributes to the role of DAG as a bi-directional regulator of platelet reactivity.

Beta 1-, beta 2- and atypical beta-adrenoceptor-mediated relaxation in rat isolated aorta.

beta-adrenoceptor-mediated relaxation was investigated in ring preparations of rat isolated thoracic aorta. Rings were pre-constricted with a sub-maximal concentration of noradrenaline (1 microM) and relaxant responses to cumulative concentrations of beta-adrenoceptor agonists obtained. The concentration-response curve (CRC) to isoprenaline was shifted to the right by propranolol (0.3 microM) with a steepening of the slope. Estimation of the magnitude of the shift from EC(50) values gave a pA(2) of 7.6. Selective beta(1)- and beta(2)-adrenoceptor antagonists, CGP 20712A (0.1 microM) and ICI 118551 (0.1 microM), respectively, produced 4 and 14 fold shifts of the isoprenaline CRC. Atypical beta-adrenoceptor agonists also produced concentration-dependent relaxation of aortic rings. The order of potency of the beta-adrenoceptor agonists was (-log EC(50)): isoprenaline (6. 25)>cyanopindolol (5.59)>isoprenaline+propranolol (5.11)>CGP 12177A (4.40)>ZD 2079 (4.24)>ZM 215001 (4.07)>BRL 37344 (3.89). Relaxation to CGP 12177A and ZM 215001 was unaffected by propranolol (0.3 microM). SR 59230A (

Relaxation to bradykinin in bovine pulmonary supernumerary arteries can be mediated by both a nitric oxide-dependent and -independent mechanism.

1. The aim of the present study was to determine the relative contribution of prostanoids, nitric oxide and K(+) channels in the bradykinin-induced relaxation of bovine pulmonary supernumerary arteries. 2. In endothelium-intact, but not denuded rings, bradykinin produced a concentration-dependent relaxation (pEC(50), 9.6+/-0.1), which was unaffected by the cyclo-oxygenase inhibitor indomethacin. The nitric oxide scavenger hydroxocobalamin (200 micro M, pEC(50), 8.5+/-0.2) and the nitric oxide synthase inhibitor L-NAME (100 micro M, pEC(50), 8.9+/-0.1) and the combination of L-NAME and hydroxocobalamin (pEC(50), 8.1+/-0.2) produced rightward shifts in the bradykinin concentration response curve. 3. The guanylyl cyclase inhibitor ODQ (10 micro M, pEC(50), 9.6+/-0.4) did not affect the response to bradykinin. 4. Elevating the extracellular [K(+)] to 30 mM did not affect the response to bradykinin but abolished the response when ODQ or L-NAME was present. 5. The K(+) channel blocker apamin (100 nM), combined with charybdotoxin (100 nM), produced a small reduction in the maximum response to bradykinin but they abolished the response to bradykinin when ODQ, L-NAME or hydroxocobalamin were present. Apamin (100 nM) combined with iberiotoxin (100 nM) also reduced the response to bradykinin in the presence of hydroxocobalamin or L-NAME. 6. The concentration response curve for sodium nitroprusside-induced relaxation was abolished by ODQ (10 micro M) and shifted to the right by apamin and charybdotoxin. 7. These studies suggest that in bovine pulmonary supernumerary arteries bradykinin can stimulate the formation of nitric oxide and activate an EDHF-like mechanism and that either of these pathways alone can mediate the bradykinin-induced relaxation. In addition nitric oxide, acting through guanylyl cyclase, can activate an apamin/charbydotoxin-sensitive K(+) channel in this tissue.

V-shaped cushion at the origin of bovine pulmonary supernumerary arteries: structure and putative function.

This study investigates the anatomic structure at the origin of pulmonary supernumerary arteries and their parent conventional artery. Histological examination showed that at the origin of each supernumerary artery the wall of the parent conventional artery is organized into a distinct V-shaped structure, which begins on the hilum side of each supernumerary artery as a funnel-shaped channel running into the supernumerary artery. The base of the channel is particularly thin walled. The lateral walls of the channel are composed of musculoelastic cushions that become more pronounced toward the supernumerary artery and fuse on its distal side, forming a baffle that projects over the supernumerary artery lumen. These V-shaped structures/cushions were observed with video stereo dissecting microscopy in both an open and closed state in isolated arteries in vitro. Pulmonary vasoconstriction of isolated arteries with the thromboxane A(2) mimetic U-46619 increased the number of V-shaped structures in the closed state. These studies indicate the presence of a novel anatomic structure at the origin of pulmonary supernumerary arteries, which may be able to regulate blood flow into the supernumerary artery.

Bounce-by-bounce cavity ring-down spectroscopy: femtosecond temporal imaging.

A time microscope (100× magnification) allows light pulses exiting an optical cavity to be viewed one at a time. A linearly chirped Gaussian pulse is mixed in a nonlinear crystal with the dispersed input waveform; the up-converted light is sent onto an output dispersive network. The resulting temporal image is recorded both with a streak camera and with a spectrometer.

Role of endothelium/nitric oxide in atypical beta-adrenoceptor-mediated relaxation in rat isolated aorta.

The role of endothelium in the modulation of classical and atypical beta-adrenoceptor-mediated vasorelaxation was investigated in ring preparations of rat isolated thoracic aorta. Rings were pre-constricted with a sub-maximal concentration of noradrenaline (1 microM) and relaxant responses to cumulative concentrations of beta-adrenoceptor agonists obtained. Endothelium removal or pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) or 1H-[1,2,4] oxadiazolol[4,3,-a] quinoxalin-1-one (ODQ, 10 microM) significantly reduced the relaxant effects of isoprenaline, but had less effect on relaxant responses to the atypical beta-adrenoceptor agonist, (+/-)-4-(3-t-butylamino-2-hydroxypropoxy)-benzimidazol-2-one hydrochloride (CGP 12177A). Sodium nitroprusside (3 nM) shifted the isoprenaline concentration-response curve to the left and restored the attenuated responses in the presence of L-NAME back to control levels. Sodium nitroprusside had little effect on the CGP 12177A concentration-response curve. The results show that the endothelium/nitric oxide (NO) pathway modulates beta-adrenoceptor-mediated vasorelaxation in rat aorta and that classical beta-adrenoceptors are modulated to a greater extent than atypical beta-adrenoceptors.

Regulation of sensitivity to 5-hydroxytryptamine in pulmonary supernumerary but not conventional arteries by a 5-HT(1D)-like receptor.

Bovine pulmonary supernumerary arteries are more sensitive to 5-hydroxtryptamine (5-HT) (pD(2) 6.43+/-0.25) than conventional arteries (pD(2) 5.32+/-0.16). This study investigated receptors for 5-HT in ring segments of these arteries. The 5-HT(2) receptor agonist, 2,5 dimethoxy-4-iodoamphetamine hydrobromide (DOI) constricts both arteries. The selective 5-HT(2) receptor antagonist ritanserin produced insurmountable antagonism of 5-HT concentration-response curves in both arteries, whereas the 5-HT(1B/1D) receptor antagonist N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'(5-methyl- 1,2,4-oxadiazol-3-yl[1,1,-biphenyl]-4-carboxamide hydrochloride (GR127935) produced much greater antagonism in supernumerary arteries. In rings preconstricted with 9,11-dideoxy-9, 11-methanoepoxy prostalagdin F(2alpha) (U46619) and relaxed with the adenylyl cyclase activator forskolin, the selective 5-HT(ID) receptor agonist 2-[5-[3-(4-methylsulphonylamino) benzyl-1,2, 4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine (L694247) reversed the relaxation. Concentration-response curves for L694247-induced reversal of forskolin-relaxation were antagonised by GR127935 in supernumerary (pK(B) 8.6) and conventional (pK(B) 8.4) arteries, whereas concentration-response curves to 5-HT-were less sensitive to antagonism by GR127935T and this was more obvious in conventional (pK(B) 7.6) than supernumerary (pK(B) 8.1) arteries. Neither the selective 5-HT(1D) receptor antagonist (1-(3-chlorophenyl)-4-[3, 3-diphenyl (2-(S,R) hydroxypropanyl)piperazine] hydrochloride (BRL15572) nor the 5-HT(1B) receptor antagonist (2,3,6, 7-tetrahydro-1'-methyl-5-[2'methyl-4'5-(methyl-1,2,4-oxadiazol-3-y l) biphenyl-4-carbonyl]furo[2,3-f]indole-3-spiro-4'-piperidine hydrochloride (SB224289) antagonised concentration-response curves induced by 5-HT or 5-HT(1)-receptor-selective agonists. In addition to the 5-HT(2A) receptor, 5-HT activates a GR127935-sensitive and a GR127935-insensitive receptor in these arteries. Supernumerary arteries have a greater proportion of GR127935-sensitive receptors, which display only some of the pharmacological characteristics of the cloned 5-HT(ID) receptor. It is possible that the GR127935-sensitive receptor could be a species homologue of the human 5-HT(1B) receptor that is insensitive to SB224289.

Phospholipid-induced human platelet activation: effects of calcium channel blockers and calcium chelators.

Human platelet activation (aggregation, [14C]-5HT release and TxB2 production) induced by the phospholipids, PAF and lysophosphatidic acid (LPA) was inhibited by EGTA, TMB-8 (an intracellular calcium antagonist) and by phenylalkylamine (Class II) but not 1,4-dihydropyridine (Class I) calcium channel blockers. Primary aggregation induced by PAF was selectively inhibited by phenylalkylamine (verapamil, methoxyverapamil) calcium channel blockers. Phospholipid-induced human platelet activation depends predominantly on the influx of extracellular calcium, possibly via specific receptor-operated calcium channels.

Intra- and interrater agreement with cumulative defect curves.

PURPOSE: To examine intra- and interrater agreement when analyzing cumulative defect curves. Cumulative defect (Bebié) curves provide a graphical representation of the visual field and allow a subjective classification of diffuse and localized loss. METHODS: We used 75 Humphrey 30-2 visual field tests, randomly chosen from a database of 782 fields of 113 patients with open-angle glaucoma. Cumulative defect curves were generated and randomly arranged into five sets, with each set containing the 75 curves in a unique sequence. Five raters (two experienced and three inexperienced) rated each set and classified each curve as showing diffuse loss, localized loss, both diffuse and localized loss, or no loss. The intra- and interrater agreement in rating the curves was then analyzed. RESULTS: Intrarater agreement ranged from 73.3-88.0% for perfect rater agreement, with 5 identical ratings. Agreement for experienced versus inexperienced raters gave similar results (means: 84.0% and 77.8%, respectively, for perfect agreement). Interrater agreement for each set, evaluated by the kappa statistic, was substantial for all 5 sets (0.65-0.71). Kappa values for each set were comparable for experienced and inexperienced raters (0.72-0.83 and 0.59-0.69 respectively). CONCLUSIONS: Analyzing the nature of visual field loss using the cumulative defect curve is simple to learn and provides high intrarater agreement as well as substantial interrater agreement.

Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling complement cascade activation in vitro using an activated complement serum calibration standard.

Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade.

Conjugation of Y. pestis F1-antigen to gold nanoparticles improves immunogenicity.

The efficacy of 15 nm gold nanoparticles (AuNP) coated with Yersinia pestis F1-antigen, as an immunogen in mice, has been assessed. The nanoparticles were decorated with F1-antigen using N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride coupling chemistry. Mice given AuNP-F1 in alhydrogel generated the greatest IgG antibody response to F1-antigen when compared with mice given AuNP-F1 in PBS or given unconjugated F1-antigen in PBS or alhydrogel. Compared with unconjugated F1-antigen, the IgG2a response was enhanced in mice dosed with AuNP-F1 in PBS (p<0.05) but not in mice immunised with AuNP-F1 in alhydrogel. All treatment groups developed a memory response to F1-antigen, the polarity of which was inflenced by formulation in alhydrogel. The sera raised against F1-antigen coupled to AuNPs was able to competitively bind to rF1-antigen, displacing protective macaque sera.