Should digestion assays be used to estimate persistence of potential allergens in tests for safety of novel food proteins?

Food allergies affect an estimated 3 to 4% of adults and up to 8% of children in developed western countries. Results from in vitro simulated gastric digestion studies with purified proteins are routinely used to assess the allergenic potential of novel food proteins. The digestion of purified proteins in simulated gastric fluid typically progresses in an exponential fashion allowing persistence to be quantified using pseudo-first-order rate constants or half lives. However, the persistence of purified proteins in simulated gastric fluid is a poor predictor of the allergenic status of food proteins, potentially due to food matrix effects that can be significant in vivo. The evaluation of the persistence of novel proteins in whole, prepared food exposed to simulated gastric fluid may provide a more correlative result, but such assays should be thoroughly validated to demonstrate a predictive capacity before they are accepted to predict the allergenic potential of novel food proteins

Assessment of potential cardiotoxic side effects of mitoxantrone in patients with multiple sclerosis

Previous studies showed that mitoxantrone can reduce disability progression in patients with multiple sclerosis (MS). There is, however, concern that it may cause irreversible cardiomyopathy with reduced left ventricular (LV) ejection fraction (EF) and congestive heart failure. The aim of this prospective study was to investigate cardiac side effects of mitoxantrone by repetitive cardiac monitoring in MS patients. The treatment protocol called for ten courses of a combined mitoxantrone (10 mg/m(2) body surface) and methylprednisolone therapy. Before each course, a transthoracic echocardiogram was performed to determine the LV end-diastolic diameter, the end-systolic diameter and the fractional shortening; the LV-EF was calculated. Seventy-three patients participated (32 males; age 48 +/- 12 years, range 20-75 years; 25 with primary progressive, 47 with secondary progressive and 1 with relapsing-remitting MS) who received at least four courses of mitoxantrone. Three of the 73 patients were excluded during the study (2 patients discontinued therapy; 1 patient with a previous history of ischemic heart disease developed atrial fibrillation after the second course of mitoxantrone). The mean cumulative dose of mitoxantrone was 114.0 +/- 33.8 mg. The mean follow-up time was 23.4 months (range 10-57 months). So far, there has been no significant change in any of the determined parameters (end-diastolic diameter, end-systolic diameter, fractional shortening, EF) over time during all follow-up investigations. Mitoxantrone did not cause signs of congestive heart failure in any of the patients. Further cardiac monitoring is, however, needed to determine the safety of mitoxantrone after longer follow-up times and at higher cumulative doses. Copyright (C) 2005 S. Karger AG, Basel

Differences in genotype and virulence among four multidrug-resistant <i>Streptococcus pneumoniae</i> isolates belonging to the PMEN1 clone

We report on the comparative genomics and characterization of the virulence phenotypes of four &lt;i&gt;S. pneumoniae&lt;/i&gt; strains that belong to the multidrug resistant clone PMEN1 (Spain&lt;sup&gt;23F&lt;/sup&gt; ST81). Strains SV35-T23 and SV36-T3 were recovered in 1996 from the nasopharynx of patients at an AIDS hospice in New York. Strain SV36-T3 expressed capsule type 3 which is unusual for this clone and represents the product of an in vivo capsular switch event. A third PMEN1 isolate - PN4595-T23 - was recovered in 1996 from the nasopharynx of a child attending day care in Portugal, and a fourth strain - ATCC700669 - was originally isolated from a patient with pneumococcal disease in Spain in 1984. We compared the genomes among four PMEN1 strains and 47 previously sequenced pneumococcal isolates for gene possession differences and allelic variations within core genes. In contrast to the 47 strains - representing a variety of clonal types - the four PMEN1 strains grouped closely together, demonstrating high genomic conservation within this lineage relative to the rest of the species. In the four PMEN1 strains allelic and gene possession differences were clustered into 18 genomic regions including the capsule, the blp bacteriocins, erythromycin resistance, the MM1-2008 prophage and multiple cell wall anchored proteins. In spite of their genomic similarity, the high resolution chinchilla model was able to detect variations in virulence properties of the PMEN1 strains highlighting how small genic or allelic variation can lead to significant changes in pathogenicity and making this set of strains ideal for the identification of novel virulence determinant

Derangement of a Factor Upstream of RARα Triggers the Repression of a Pleiotropic Epigenetic Network

Chromatin adapts and responds to extrinsic and intrinsic cues. We hypothesize that inheritable aberrant chromatin states in cancer and aging are caused by genetic/environmental factors. In previous studies we demonstrated that either genetic mutations, or loss, of retinoic acid receptor alpha (RARalpha), can impair the integration of the retinoic acid (RA) signal at the chromatin of RA-responsive genes downstream of RARalpha, and can lead to aberrant repressive chromatin states marked by epigenetic modifications. In this study we tested whether the mere interference with the availability of RA signal at RARalpha, in cells with an otherwise functional RARalpha, can also induce epigenetic repression at RA-responsive genes downstream of RARalpha.To hamper the availability of RA at RARalpha in untransformed human mammary epithelial cells, we targeted the cellular RA-binding protein 2 (CRABP2), which transports RA from the cytoplasm onto the nuclear RARs. Stable ectopic expression of a CRABP2 mutant unable to enter the nucleus, as well as stable knock down of endogenous CRABP2, led to the coordinated transcriptional repression of a few RA-responsive genes downstream of RARalpha. The chromatin at these genes acquired an exacerbated repressed state, or state "of no return". This aberrant state is unresponsive to RA, and therefore differs from the physiologically repressed, yet "poised" state, which is responsive to RA. Consistent with development of homozygosis for epigenetically repressed loci, a significant proportion of cells with a defective CRABP2-mediated RA transport developed heritable phenotypes indicative of loss of function.Derangement/lack of a critical factor necessary for RARalpha function induces epigenetic repression of a RA-regulated gene network downstream of RARalpha, with major pleiotropic biological outcomes

Pain control after total knee arthroplasty: a randomized trial comparing local infiltration anesthesia and continuous femoral block

Local infiltration analgesia (LIA) is a new multimodal wound infiltration method. It has attracted growing interest in recent years and is widely used all over the world for treating postoperative pain after knee and hip arthroplasty. This method is based on systematic infiltration of a mixture of ropivacaine, a long acting local anesthetic, ketorolac, a cyclooxygenase inhibitor (NSAID), and adrenalin around all structures subject to surgical trauma inknee and hip arthroplasty. Two patient cohorts of 40 patients scheduled for elective total knee arthroplasty (TKA) and 15 patients scheduled for total hip arthroplasty (THA) contributed to the work presented in this thesis. In a randomized trial the efficacy of LIA in TKA with regard to pain at rest and upon movement was compared to femoral block. Both methods result in a high quality pain relief and similar morphine consumption, but fewer patients in the LIA group reported pain of 7/10 on any occasion during the 24 h monitoring period (paper I). In the same patient cohort the maximal total plasma concentration of ropivacaine was below the established toxic threshold for most patients although a few reached potentially toxic concentrations of 1.4-1.7 mg/L. The time to maximal detected plasma concentration was around 4-6 h after release of tourniquet in TKA (paper II). All patients in the THA cohort were subjected to the routine LIA protocol. In these patients both the total and unbound plasma concentration of ropivacaine was determined. The concentration was below the established toxic threshold. As ropivacaine binds to a-1 acid glycoprotein(AAG) we assessed the possibility that increased AAG may decrease the unbound concentration of ropivacaine. A40 % increase in AAG was detected during the first 24 h after surgery, however the fraction of unbound ropivacaine remained the same. There was a trend towards increased C max of ropivacaine with increasing age and decreasing creatinine clearance but the statistical power was too low to draw any conclusion (paper III). Administration of 30mg ketorolac according to the LIA protocol both in TKA and THA resulted in a similar Cmax as previously reported after 10 mg intramuscular ketorolac (paper II, paper IV). Neither age, nor body weight or BMI, nor creatinine clearance, correlates to maximal ketorolac plasma concentration or total exposure to ketorolac (AUC) (paper IV). In conclusion, LIA provides good postoperative analgesia which is similar to femoral block after total knee arthroplasty. The plasma concentration of ropivacaine seems to be below toxic levels in most TKA patients. The unbound plasma concentration of ropivcaine in THA seems to be below the toxic level. The use of ketorolac in LIA may not be safer than other routes of administration, and similar restrictions should be applied in patients at risk of developing side effects

Phase I study of the combination of losoxantrone and cyclophosphamide in patients with refractory solid tumours

Losoxantrone is a DNA intercalator that was developed with the potential to replace anthracyclines. The recommended single agent dose of losoxantrone is 50 mg m−2 every 3 weeks. We conducted a phase I study of losoxantrone and a fixed dose of cyclophosphamide on a q3 weekly schedule. Forty-nine patients were enrolled, of which 46 were evaluable for toxicity. The dose-limiting toxicity was neutropenia at the maximum tolerable losoxantrone dose of 45 mg m−2. With granulocyte colony-stimulating factor support, significant further dose escalation of losoxantrone was achieved. Cardiotoxicity was seen with cumulative dosing. Pharmacokinetics of losoxantrone revealed linear kinetics and triphasic clearance, with significant interpatient variability. No objective responses were seen in this study. Neutropenia was dose-limiting in this combination with or without granulocyte colony-stimulating factor support. The recommended dose for further testing is cyclophosphamide 500 mg m−2 followed by losoxantrone 95 mg m−2 with granulocyte colony-stimulating factor support

Acupuncture in the treatment of rheumatoid arthritis: a double-blind controlled pilot study

<p>Abstract</p> <p>Background</p> <p>In planning a randomized controlled trial of acupuncture, we conducted a pilot study using validated outcome measures to assess the feasibility of the protocol, and to obtain preliminary data on efficacy and tolerability of 3 different forms of acupuncture treatment as an adjunct for the treatment of chronic pain in patients with Rheumatoid arthritis (RA).</p> <p>Methods</p> <p>The study employs a randomized, prospective, double-blind, placebo-controlled trial to evaluate the effect of electroacupuncture (EA), traditional Chinese acupuncture (TCA) and sham acupuncture (Sham) in patients with RA. All patients received 20 sessions over a period of 10 weeks. Six acupuncture points were chosen. Primary outcome is the changes in the pain score. Secondary outcomes included the changes in the ACR core disease measures, DAS 28 score and the number of patients who achieved ACR 20 at week 10.</p> <p>Results</p> <p>From 80 eligible patients, 36 patients with mean age of 58 ± 10 years and disease duration of 9.3 ± 6.4 years were recruited. Twelve patients were randomized to each group. Twelve, 10 and 7 patients from the EA, TCA and Sham group respectively completed the study at 20 weeks (p < 0.03); all except one of the premature dropouts were due to lack of efficacy. At week 10, the pain score remained unchanged in all 3 groups. The number of tender joints was significantly reduced for the EA and TCA groups. Physician's global score was significantly reduced for the EA group and patient's global score was significantly reduced for the TCA group. All the outcomes except patient's global score remained unchanged in the Sham group.</p> <p>Conclusion</p> <p>This pilot study has allowed a number of recommendations to be made to facilitate the design of a large-scale trial, which in turn will help to clarify the existing evidence base on acupuncture for RA.</p> <p>Trial registration</p> <p>ClinicalTrials.gov NCT00404443</p

Structural Basis for Certain Naturally Occurring Bioflavonoids to Function as Reducing Co-Substrates of Cyclooxygenase I and II

Recent studies showed that some of the dietary bioflavonoids can strongly stimulate the catalytic activity of cyclooxygenase (COX) I and II in vitro and in vivo, presumably by facilitating enzyme re-activation. In this study, we sought to understand the structural basis of COX activation by these dietary compounds.A combination of molecular modeling studies, biochemical analysis and site-directed mutagenesis assay was used as research tools. Three-dimensional quantitative structure-activity relationship analysis (QSAR/CoMFA) predicted that the ability of bioflavonoids to activate COX I and II depends heavily on their B-ring structure, a moiety known to be associated with strong antioxidant ability. Using the homology modeling and docking approaches, we identified the peroxidase active site of COX I and II as the binding site for bioflavonoids. Upon binding to this site, bioflavonoid can directly interact with hematin of the COX enzyme and facilitate the electron transfer from bioflavonoid to hematin. The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity. Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation