High fludarabine exposure and relationship with treatment-related mortality after nonmyeloablative hematopoietic cell transplantation.

Despite its common use in nonmyeloablative preparative regimens, the pharmacokinetics of fludarabine are poorly characterized in hematopoietic cell transplantation (HCT) recipients and exposure-response relationships remain undefined. The objective of this study was to evaluate the association between plasma F-ara-A exposure, the systemically circulating moiety of fludarabine, and engraftment, acute GVHD, TRM and OS after HCT. The preparative regimen consisted of CY 50 mg/kg/day i.v. day -6; plus fludarabine 30-40 mg/m²/day i.v. on days -6 to -2 and TBI 200 cGy on day -1. F-ara-A pharmacokinetics were carried out with the first dose of fludarabine in 87 adult patients. Median (range) F-ara-A area-under-the-curve (AUC((0-∞))) was 5.0 μg h/mL (2.0-11.0), clearance 15.3 L/h (6.2-36.6), C(min) 55 ng/mL (17-166) and concentration on day(zero) 16.0 ng/mL (0.1-144.1). Despite dose reductions, patients with renal insufficiency had higher F-ara-A exposures. There was strong association between high plasma concentrations of F-ara-A and increased risk of TRM and reduced OS. Patients with an AUC((0-∞)) greater than 6.5 μg h/mL had 4.56 greater risk of TRM and significantly lower OS. These data suggest that clinical strategies are needed to optimize dosing of fludarabine to prevent overexposure and toxicity in HCT

Production of Recombinant Peanut Allergen Ara h 2 using Lactococcus lactis

<p>Abstract</p> <p>Background</p> <p>Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high-level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium <it>Lactococcus lactis </it>is an attractive microorganism for use in the production of protein therapeutics. <it>L. lactis </it>is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using <it>L. lactis</it>.</p> <p>Results</p> <p>A synthetic ara h 2 gene was cloned into an <it>L. lactis </it>expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N-terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2.</p> <p>Conclusion</p> <p>Recombinant production of Ara h 2 using <it>L. lactis </it>can offer high yields of secreted, full length and immunologically active allergen. The <it>L. lactis </it>expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics.</p

L-arabinose co-ingestion delays glucose absorption derived from sucrose in healthy men and women : A double-blind, randomized crossover trial

Dietary interventions to delay carbohydrate digestion or absorption can effectively prevent hyperglycaemia in the early postprandial phase. L-arabinose can specifically inhibit sucrase. It remains to be assessed whether co-ingestion of L-arabinose with sucrose delays sucrose digestion, attenuates subsequent glucose absorption and impacts hepatic glucose output. In this double-blind, randomised crossover study, we assessed blood glucose kinetics following ingestion of a 200-ml drink containing 50 g of sucrose with 7·5 g of L-arabinose (L-ARA) or without L-arabinose (CONT) in twelve young, healthy participants (24 ± 1 years; BMI: 22·2 ± 0·5 kg/m2). Plasma glucose kinetics were determined by a dual stable isotope methodology involving ingestion of (U-13C6)-glucose-enriched sucrose, and continuous intravenous infusion of (6,6–2H2)-glucose. Peak glucose concentrations reached 8·18 ± 0·29 mmol/l for CONT 30 min after ingestion. In contrast, the postprandial rise in plasma glucose was attenuated for L-ARA, because peak glucose concentrations reached 6·62 ± 0·18 mmol/l only 60 min after ingestion. The rate of exogenous glucose appearance for L-ARA was 67 and 57 % lower compared with CONT at t = 15 min and 30 min, respectively, whereas it was 214 % higher at t = 150 min, indicating a more stable absorption of exogenous glucose for L-ARA compared with CONT. Total glucose disappearance during the first hour was lower for L-ARA compared with CONT (11 ± 1 v. 17 ± 1 g, P < 0·0001). Endogenous glucose production was not differentially affected at any time point (P = 0·27). Co-ingestion of L-arabinose with sucrose delays sucrose digestion, resulting in a slower absorption of sucrose-derived glucose without causing adverse effects in young, healthy adults

Stability of Transgene Expression in Reduced Allergen Peanut (\u3ci\u3eArachis hypogaea\u3c/i\u3e L.) across Multiple Generations and at Different Soil Sulfur Levels

Transgenic peanut (Arachis hypogaea L.) containing a gene designed for RNA interference (RNAi) showed stable complete silencing of Ara h 2 and partial silencing of Ara h 6, two potent peanut allergens/proteins, along with minimal collateral changes to other allergens, Ara h 1 and Ara h 3, across three generations (T3, T4, and T5) under field conditions. Different soil sulfur levels (0.012, 0.3, and 3.0 mM) differentially impacted sulfur-rich (Ara h 2, Ara h 3, and Ara h 6) versus sulfur-poor (Ara h 1) proteins in non-transgenic versus transgenic peanut. The sulfur level had no effect on Ara h 1, whereas low sulfur led to a significant reduction of Ara h 3 in transgenic and non-transgenic seeds and Ara h 2 and Ara h 6 in non-transgenic but not in transgenic peanuts because these proteins already were reduced by gene silencing. These results demonstrate stability of transgene expression and the potential utility of RNAi in allergen manipulation

KANDUNGAN TERPENOID DALAM DAUN ARA (Ficus carica L.) SEBAGAI AGEN ANTIBAKTERI TERHADAP BAKTERI Methicillin-Resistant Staphylococcus aureus

ABSTRACT Fig leaves (Ficus carica L.) are known to the public with many health benefits. The content of efficacious compounds in fig leaves such as terpenoids has potential as an antibacterial and needs to be known. This study aims to determine the antibacterial activity of terpenoid content in extracts and fractions of fig leaves (Ficus carica L.) on the growth of Methicillin-Resistant Staphylococcus aureus (MRSA) bacteria by contact bioautography. Extraction was carried out by stratified soxhletation with n-hexane and ethyl acetate. Separation was carried out by coloum vacuum liquid chromatography (VLC) method. The wells diffusion method is used as the antibacterial activity test, while the TLC contact bioautography test is carried out to determine the antibacterial activity of the terpenoid content in the extracts and fractions. Extracts of n-hexane, ethyl acetate, and ethanol of fig leaf (Ficus carica L.) have antibacterial activity against MRSA with a diameter of inhibitory zone 0.111 ± 0.003; 0.328 ± 0.026, 1.044 ± 0.115 cm, and show significant differences. Extracts of n-hexane, ethyl acetate, and ethanol of fig leaf (Ficus carica L.) contain terpenoids. The fraction of ethyl acetate and ethanol extracts of fig leaves (Ficus carica L.) contains terpenoid compounds which can provide antibacterial activity against MRSA by TLC contact bioautography. Keywords:       fig leaves, Ficus carica L., antibacterial, Methicillin-Resistant S. aureus.  ABSTRAK Daun ara (Ficus carica L.) dikenal masyarakat dengan banyak manfaat dalam bidang kesehatan. Kandungan senyawa berkhasiat dalam daun ara seperti terpenoid berpotensi sebagai antibakteri dan perlu diketahui. Penelitian ini bertujuan untuk mengetahui aktivitas antibakteri kandungan terpenoid dalam ekstrak maupun fraksi daun ara (Ficus carica L.) terhadap pertumbuhan bakteri Methicillin-Resistant Staphylococcus aureus (MRSA) secara KLT bioautografi kontak. Ekstraksi dilakukan dengan cara soxhletasi bertingkat dengan pelarut n-heksana dan etil asetat. Pemisahan dilakukan dengan metode kromatografi kolom vakum cair (KVC). Uji aktivitas antibakteri menggunakan metode difusi sumuran, sedangkan uji bioautografi kontak dilakukan untuk mengetahui aktivitas antibakteri kandungan terpenoid dalam ekstrak dan fraksi. Ekstrak n-heksana, etil asetat, dan etanol daun ara (Ficus carica L.) mempunyai aktivitas antibakteri terhadap MRSA dengan diameter zona hambat berturut-turut 0,111±0,003; 0,328±0,026, 1,044±0,115 cm, dan menunjukkan perbedaan signifikan. Ekstrak n-heksana, etil asetat, dan etanol daun ara (Ficus carica L.) mengandung terpenoid. Fraksi dari ekstrak etil asetat dan etanol daun ara (Ficus carica L.) mengandung senyawa terpenoid yang dapat memberikan aktivitas antibakteri terhadap MRSA secara KLT bioautografi kontak. Kata kunci : daun ara, Ficus carica L., antibakteri, Methicillin-Resistant S. aureu

Effect of N-arachidonoyl-l-serine on human cerebromicrovascular endothelium

AbstractN-arachidonoyl-l-serine (ARA-S) is an endogenous lipid, chemically related to the endocannabinoid, N-arachidonoyl ethanolamine (i.e., anandamide) and with similar physiologic and pathophysiologic functions. Reports indicate that ARA-S possesses vasoactive and neuroprotective properties resembling those of cannabinoids. However, in contrast to cannabinoids, ARA-S binds weakly to its known classical receptors, CB1 and CB2, and is therefore considered to be a ‘cannabinoid-like’ substance. The originally described ARA-S induced-endothelial-dependent vasorelaxation was not abrogated by CB1, CB2 receptor antagonists or TRPV1 competitive inhibitor. The present report demonstrates that ARA-S enhances the fluorescence staining of both cannabinoid receptors (CB1 and CB2) in human brain endothelial cells (HBEC). This reaction is specific since it was reduced by respective selective receptor antagonist (SR141716A and SR141728A). ARA-S alone or in the presence of ET-1 was shown to alter the cytoskeleton (actin). Both ARA-S stimulated phosphorylation of various kinases (MAPK, Akt, JNK and c-JUN) and alteration of cytoskeleton are mediated via CB1, CB2 and TRPV1 receptors. The findings also showed the involvement of Rho/Rock and PI3/Akt/NO pathways in the ARA-S-induced phosphorylation of kinases and actin reorganization in HBEC. All of the above mentioned ARA-S-induced effects were reduced by the treatment with LY294002 (inhibitor of PI3/Akt kinase), except MAPK kinase. In addition, MAPK, JNK, c-JUN phosphorylation were inhibited by H1152 (inhibitor of Rho/ROCK kinase), except Akt kinase. Furthermore, PI3/Akt pathway was inhibited by pretreatment with l-NAME (inhibitor of NOS). The findings suggest that ARA-S is a modulator of Rho kinase and may play a critical role in the regulation of its activity and subsequent effects on the cytoskeleton and its role in supporting essential cell functions like vasodilation, proliferation and movement

Growth survival and resistance to hypersaline stress in larval black sea bass (Centropristis striata) fed varying levels of dietary arachidonic acid (20:4n-6)

Significant advances have been made in controlled breeding and knowledge of environmental requirements for culture of larval black sea bass (Centropristis striata), but there still is relatively little published data on the nutritional requirements of the larval stages, including optimal methods for live prey enrichment with essential fatty acids. The objectives of this study were to determine the arachidonic acid (ARA, 20:4n-6) requirements of black sea bass larvae from the first feeding through metamorphic stages approximately 24 days post-hatching (d24ph). Thirty 15-L aquaria were stocked with d1ph yolksac stage larvae at 100 ind./L. Salinity (34 g/L), temperature (22 °C), photoperiod (18L: 6D), light intensity (1,000 lux), diffused aeration (100 mL/min) and D.O. (&gt; 5 mg/L) were held constant. Background microalgae Nannochloropsis oculata was added daily to maintain 300,000 cells/L. To determine ARA requirements of larvae, live prey organisms, rotifers (Brachionus sp.) and Artemia were enriched with emulsions containing 10% docosahexaenoic acid, DHA (22:6n-3) and five different levels of ARA (0, 6, 8, 10 and 12% total fatty acids, TFA). In a sixth treatment, live prey was enriched with Algamac 2000 (26% DHA, 0% ARA), a commercial fatty acid booster. Rotifers were fed from d2ph at 10 ind./mL, increasing to 23 ind./mL by d18ph. On d18ph Artemia were fed at 0.5 ind./mL, increasing to 3 ind./mL on d22ph. Rotifer feeding ceased on d20ph. Larvae were sampled on d4, d10, d17 and d24ph to monitor survival and growth (NL, wet wt. and dry wt.). On d24ph, hypersaline (55 g/L) stress resistance (ST-50) was evaluated. To measure Na,K,ATP-ase expression, salinity was increased to a sublethal level of 42 g/L and larvae were sampled at 0 h and after 24 h for mRNA analysis by quantitative real-time PCR (qRT-PCR). On d24ph, larval fatty acid profiles reflected dietary levels. Larval NL, wet wt. and dry wt. increased steadily in all treatments during the study, with no significant (P &gt; 0.05) treatment effects. On d24ph, no significant treatment differences in survival (range = 24.3-32.7%) or hypersaline stress (range = 27.1-31.8) resistance were evident. However, larvae fed diets supplemented with ARA (6-12% TFA) demonstrated a significant (P &lt; 0.05) increase in relative mRNA expression of Na+ K+ ATPase after 24 h, whereas larvae fed 0% ARA and Algamac showed no increase. The results indicate that dietary supplementation with ARA at 6-12% promoted the adaptive physiological responses to hypersalinity stress and hypo-osmoregulatory ability in black sea bass larvae

Utility of Specific IgE to Ara h 2 in Italian Allergic and Tolerant Children Sensitized to Peanut

Emerging data suggest that measurement of serum IgE to peanut components can be clinically helpful and more accurate than IgE to whole peanut to predict peanut allergy. Not all studies have used prospective samples, multiple components and oral challenges. Currently, there are no data on this topic involving Italian children. 32 patients (23 males; median age 9 years) with reported history for peanut allergy and evidence of peanut sensitization (skin prick test to peanut extract ≥ 3mm) have been analyzed for serum IgE to whole peanut and recombinant allergen components Ara h 1, 2, 3, 8, and 9 with Immuno CAP and completed an open oral food challenge with peanut. 12 (37.5%) children had a positive challenge to peanut and were considered allergic. No differences were seen between the median values of IgE to peanut, Ara h 1, 3, 8 and 9 in allergic and tolerant children to peanut challenge. Noteworthy, 5 of 20 tolerant children had IgE to peanut> 15 kUA/l which is commonly considered a predictive value of peanut allergy. Conversely, a significant difference was seen when comparing the median value of IgE to Ara h 2 in the two groups: 0.75 kUA/l (IQR: 0.22-4.34 kUA/l) in allergic children versus 0.1 kUA/l (IQR: 0.1-0.12 kUA/l) in tolerant ones (P< 0.001). IgE levels to Ara h 2 are significantly higher in children that react to oral peanut challenge. Our findings in Italian children have been in line with recent reports in various populations of Northern Europe, the US and Australia and add confirmatory evidence that analysis of IgE to Ara h 2 could reduce the need for peanut challenge in suspected allergic patients