Oxidized quercetin reacts with thiols rather than with ascorbate: implication for quercetin supplementation

When an antioxidant scavenges a reactive species, i.e., when it exerts its antioxidant activity, the antioxidant is converted into potentially harmful oxidation products. In this way, the antioxidant quercetin might yield an ortho-quinone, denoted as QQ, which has four tautomeric forms, i.e., the ortho-quinone and three quinonmethides. We evaluated the interaction of QQ with ascorbate or glutathione (GSH). Ascorbate recycles QQ to the parent compound quercetin, while GSH forms two adducts with QQ, i.e., 6-GSQ and 8-GSQ. When both GSH and ascorbate are present, QQ is converted exclusively into GSQ. In the absence of GSH, protein thiols will be arylated by QQ. This protein arylation is not prevented by ascorbate. Thiol arylation by quinones and quinonmethides can impair several vital enzymes. This implies that the product formed when quercetin displays its antioxidant scavenging effect is toxic in the absence of GSH. Therefore, an adequate GSH level should be maintained when quercetin is supplemented.     

Knockout of Kir6.2 negates ischemic preconditioning-induced protection of myocardial energetics

Although ischemic preconditioning induces bioenergetic tolerance and thereby remodels energy metabolism that is crucial for postischemic recovery of the heart, the molecular components associated with preservation of cellular energy production, transfer, and utilization are not fully understood. Here myocardial bioenergetic dynamics were assessed by (18)O-assisted (31)P-NMR spectroscopy in control or preconditioned hearts from wild-type (WT) or Kir6.2-knockout (Kir6.2-KO) mice that lack metabolism-sensing sarcolemmal ATP-sensitive K(+) (K(ATP)) channels. In WT vs. Kir6.2-KO hearts, preconditioning induced a significantly higher total ATP turnover (232 +/- 20 vs. 155 +/- 15 nmol x mg protein(-1) x min(-1)), ATP synthesis rate (58 +/- 3 vs. 46 +/- 3% (18)O labeling of gamma-ATP), and ATP consumption rate (51 +/- 4 vs. 31 +/- 4% (18)O labeling of P(i)) after ischemia-reperfusion. Moreover, preconditioning preserved cardiac creatine kinase-catalyzed phosphotransfer in WT (234 +/- 26 nmol x mg protein(-1) x min(-1)) but not Kir6.2-KO (133 +/- 18 nmol x mg protein(-1) x min(-1)) hearts. In contrast with WT hearts, preconditioning failed to preserve contractile recovery in Kir6.2-KO hearts, as tight coupling between postischemic performance and high-energy phosphoryl transfer was compromised in the K(ATP)-channel-deficient myocardium. Thus intact K(ATP) channels are integral in ischemic preconditioning-induced protection of cellular energetic dynamics and associated cardiac performance.     

Apolipoprotein A-I, an antimicrobial protein in Oncorhynchus mykiss: evaluation of its expression in primary defence barriers and plasma levels in sick and healthy fish

Antimicrobial proteins and peptides play an important role in the primary defence barriers in vertebrates and invertebrates. In a previous study it was shown that high-density lipoprotein (HDL) and its major apolipoproteins, ApoA-I and ApoA-II display antimicrobial activity in the carp (Cyprinus carpio L.). The aim of this study was to evaluate if ApoA-I conserves this defensive function in a salmonid fish like the rainbow trout, in spite of the low level of primary sequence conservation between fish ApoA-I. Here it is shown that trout ApoA-I displays an antimicrobial activity in the micromolar range against Gram positive and Gram negative bacteria, including some fish pathogens. In addition, its expression was also demonstrated by immunohistochemistry and RT-PCR in epidermis, gills and intestinal mucosa, which constitute the main primary defence barriers in fish. Finally, no significant difference in the hepatic expression and plasma levels of this abundant apolipoprotein was found in groups of healthy and diseased fish, in clear contrast with mammals where ApoA-I have been considered a negative acute phase reactant. These findings suggest that ApoA-I could constitute an important innate immunity effector in trout and perhaps other teleost fish.     

Role of reactive oxygen species in intestinal diseases.

It is well known that reactive oxygen metabolites are generated during several pathologies, and that they are able to disturb many cellular processes and eventually lead to cellular injury. After intestinal ischemia, reactive oxygen species are produced when the ischemic tissue is reperfused. The enzyme xanthine oxidase is thought to play a key role in this process. As a result of this oxygen radical production, the permeability of the endothelium and the mucosa increases, allowing infiltration of inflammatory leukocytes into the ischemic area. Moreover, reactive oxygen species are also indirectly involved in leukocyte activation. In turn, these inflammatory cells respond with the production of oxygen radicals, which play an important role in the development of tissue injury. Thus, intestinal ischemia and reperfusion evokes an inflammatory response. Also during chronic intestinal inflammatory diseases, reactive oxygen metabolites are proposed to play an important role in the pathology. Scavenging of reactive oxygen species will thus be beneficial in these disorders.     

Heterodimeric complex formation with CD8 and TCR by bispecific antibody sustains paracrine IL-2-dependent growth of CD3+ CD8+ T cells.

During physiologic activation of mature CD8+ T cells, TCR and CD8 bind to the same Ag-complexed MHC class I molecule. Thereby, close proximity is induced between CD8 and the TCR/CD3 complex. During this engagement, CD8 may deliver TCR-independent signals via its associated protein tyrosine kinase, p56lck. We studied the potential biologic effects of close association between CD8 and TCR/CD3 complexes by using a bispecific antibody (bsAb) directed against both TCR and CD8 molecules. This hybrid hybridoma (quadroma)-produced bsAb binds as a monomeric molecule to CD3+ CD8+ but not CD3+ CD4+ T cells. The bsAb proved capable of inducing the cytotoxic effector function of cloned CD3+ CD8+ T cells but not of CD3+ CD4+ T cells. When the bsAb was presented to resting T cells by monocytes, proliferation of the CD3+ CD4+ but not the CD3+ CD8+ subset of T lymphocytes was induced. Parental anti-TCR antibody induced vigorous growth of cells of both subsets. Essentially identical results were obtained when bsAb was presented in an immobilized fashion. The unresponsiveness of the CD3+ CD8+ T cells with respect to mitogenesis could be restored by exogenous rIL-2. The data suggest that bsAb-induced activation differs from activation by monospecific anti-TCR antibody. The former appears to more closely mimic physiologic Ag-induced signaling, because it leads to a similar paracrine IL-2-dependent growth pattern. The bsAb may, therefore, be instrumental in studying T cell signaling pathways, in particular the role of CD8-associated p56lck therein.     

CD8 T cell activation after intravenous administration of CD3×CD19 bispecific antibody in patients with non-Hodgkin lymphoma

A bispecific antibody directed to T and B cells (CD3×CD19 bsAb) was daily infused intravenously in escalating doses from 10 g up to 5 mg in three patients with chemotherapy-resistant non-Hodgkin lymphoma; in this way we aimed to activate T cells to kill the malignant B cells. Only limited toxicity was observed, consisting of moderate fever preceded by chills or shivers and mild thrombocytopenia. No human anti-(mouse Ig) antibodies were found. Pharmacokinetics showed at _1/2 of 10.5 h with peak levels of 200–300 ng/ml after infusion of 2.5 mg bsAb. bsAb in serum was functionally active in vitro. After bsAb infusion a rise in serum tumour necrosis factor was observed, accompanied by an increase in soluble CD8 and to some extent in soluble interleukin-2 receptor (IL-2R), but not in interferon , IL-4 or soluble CD4. No evidence was found for monocyte activation (no increases in IL-6, IL-8 or IL-1ß in serum). No gross changess in histology or number of IL-2R⁺, CD4⁺ or CD8⁺ cells were found in the lymph nodes after therapy, but one patient showed activated CD8⁺ T cells within the tumour nodules. In conclusion, after intravenously administered CD3×CD19 bsAb only moderate toxicity was found, probably due to CD8⁺ T cell activation and cytokine release, without CD4⁺ T cell activation.     

Histamine as a marker for hydroxyl radicals

During inflammation an influx of neutrophils and release of mediators from mast cells (such as histamine) take place. The stimulated neutrophils can produce reactive oxygen species (ROS). One of these ROS is the highly reactive hydroxyl radical (OH(.)). It would be interesting to be able to quantify the extent of ROS formation. We investigated if histamine which is present at the inflammation site can serve as an endogenous marker for the formation of OH(.). We found that histamine after incubation with OH(.) gave two distinct products in our HPLC system. One of the products gave the same characteristics as the synthesized 2-imidazolone derivative of histamine. This suggests that this derivative will be formed when histamine is incubated with OH(.).     

CA 125: the past and the future

Over the last 15 years, substantial progress has been made in understanding the potential and the limitations of the CA 125 assay. More than 2000 papers have been published concerning laboratory and clinical studies of CA 125. The original CA 125 assay utilized the OC 125 antibody that recognizes the CA 125 epitope on a high molecular weight glycoprotein. Despite repeated attempts, the gene encoding the peptide component has not yet been cloned. Monoclonal antibodies have been raised against other epitopes expressed by this molecule, leading to the development of the CA 125-II assay that exhibits less day-to-day variation. Using either assay, elevated levels of CA 125 are detected in a number of benign conditions, including endometriosis. CA 125 is most consistently elevated in epithelial ovarian cancer, but can be expressed in a number of gynecologic (endometrial, fallopian tube) and non-gynecologic (pancreatic, breast, colon and lung) cancers. The best established application of the CA 125 assay is in monitoring ovarian cancer. The rate of decline in CA 125 during primary chemotherapy has been an important independent prognostic factor in several multivariate analyses. Persistent elevation of CA 125 at the time of a second look surgical surveillance procedure predicts residual disease with > 95% specificity. Rising CA 125 values have preceded clinical detection of recurrent disease by at least 3 months in most, but not all studies. Given the modest activity of salvage chemotherapy, this information has not yet impacted on survival. Rising CA 125 during subsequent chemotherapy has been associated with progressive disease in more than 90% of cases. CA 125 may serve as an effective surrogate marker for clinical response in phase II trials of new drugs. CA 125 levels can aid in distinguishing malignant from benign pelvic masses, permitting effective triage of patients for primary surgery. Early detection of ovarian cancer remains the most promising application of CA 125. An algorithm has been developed that estimates the risk of ovarian cancer (ROC) based upon the level and trend of CA 125 values. A major trial has been initiated that uses the ROC algorithm to trigger transvaginal sonography and/or subsequent laparotomy. Such a trial could demonstrate improvement in survival through early detection. This strategy should provide adequate specificity, but sensitivity for early stage disease may not be optimal. In the future, improved sensitivity may be attained using multiple markers and neural network analysis. Most serum tumor markers have been proteins or carbohydrates, but lipid markers such as lysophosphatidic acid deserve evaluation. Genomic and proteonomic technologies should identify additional novel markers.