Pitfalls in anti-influenza T cell detection by Elispot using thimerosal containing pandemic H1N1 vaccine as antigen.

Monitoring T cells in combination with humoral response may be of value to predict clinical protection and cross-protective immunity after influenza vaccination. Elispot technique which measures cytokine produced after antigen-specific T cell stimulation is used routinely to detect and characterize anti-viral T cells. We found that the preservative thimerosal present in most H1N1 pandemic vaccines, induced in vitro abortive activation of T cells followed by cell death leading to false-positive results with the Elispot technique. The size of the spots, usually not measured in routine analysis, appears to be a discriminative criterion to detect this bias. Multi-dose vials of vaccine containing thimerosal remain important for vaccine delivery and our results alert about false-positive results of Elispot to monitor the clinical efficacy of these vaccines. We showed that this finding extends for other T cell monitoring techniques based on cytokine production such as ELISA. Although measuring in vitro immune response using the whole vaccine used for human immunization directly reflects in vivo global host response to the vaccine, the present study strongly supports the use of individual vaccine components for immune monitoring due to the presence of contaminants, such as thimerosal, leading to a bias in interpretation of the results.

Audiological findings in 100 USH2 patients.

Bilateral sensorineural hearing loss (HL), classically described as mild to severe with a typically down-sloping audiometric configuration, is the earliest symptom occurring in Usher syndrome type II (USH2). Audiological findings were analyzed in a total of 100 USH2 patients (92 families) divided into three groups according to the gene involved: 88 USH2A, 10 GPR98 and 2 DFNB31 patients. A fine analysis of audiograms was performed (pure tone average, degree of severity, configuration). The median age of HL diagnosis was 5 years (range 8 months-31 years) although the median age at USH2 diagnosis was 34.5 (range 8-76). Moderate HL was predominant (76%) and a gently down-sloping configuration characterized most audiograms (66%). No statistically significant difference was found between USH2A and GPR98 patients but a tendency was clearly noted for more GPR98 patients to present with severe hearing loss. It is not possible to predict the mutated gene from audiograms.

[Myocardial infarction and ovarian stimulation: case report]. / Infarctus du myocarde et stimulation ovarienne : à propos d'un cas.

Female infertility treated by ovarian stimulation can lead to arterial thrombosis particularly when ovarian hyperstimulation syndrome emerges. Myocardial infarction have been reported thrice, in one case even before artificial ovulation induction. A 25-year-old female with primary infertility underwent ovarian stimulation and eight days after ovulation induction and intra-uterine insemination suffered from a troponin positive non-ST-elevation myocardial infarction of the inferior wall. Coronary angiogram was normal and contrast-enhanced cardiovascular magnetic resonance imaging confirmed the subendocardial inferior infarct. This protocol included sole triptorelin administration followed by 23 recombinant follicle stimulating hormone injections and concluded by recombinant choriogonadotrophin. There was no ovarian hyperstimulation syndrome. Large biological screening did not retrieve any predisposition for arterial thrombosis. Clinical outcome was excellent. Despite weak causal link, we emphasize that chest pain during ovarian stimulation protocol should rise clinical concern for acute coronary syndrome.

Soluble interleukin-2 receptor and metalloproteinase-9 expression in head and neck cancer: prognostic value and analysis of their relationships.

In a series of 84 head and neck patients, a statistically significant correlation was observed between high serum soluble interleukin (IL)-2 receptor alpha (sIL-2Ralpha) (P = 0.034) and metalloproteinase-9 (MMP-9) concentrations (P = 0.036) at diagnosis and a shorter survival of these patients. As MMP-9 has been shown to mediate cleavage of IL-2Ralpha (CD25) by preactivated T cells, we looked for a relationship between MMP-9 expression and soluble IL-2Ralpha serum concentrations in these cancer patients. We did not find any correlation between intratumoral expression of MMP-9 or serum MMP-9 concentrations and serum sIL-2Ralpha levels. These results led us to reassess the role of MMP-9 in the release of sIL-2Ralpha. Treatment of Kit225 leukaemic cells with recombinant MMP-9 slightly decreased membrane CD25 expression and was associated with an increased concentration of sIL-2Ralpha in the supernatants. However, using a selective inhibitor of MMP-9 we did not succeed in specifically inhibiting the release of sIL-2Ralpha by the Kit225 cell line or by phytohaemagglutinin (PHA)-activated peripheral blood mononuclear cells. In addition, in a preclinical mouse model, basal serum sIL-2Ralpha concentrations and sIL-2Ralpha production by activated cells were not altered in MMP-9-deficient mice compared to wild-type mice. Interestingly, a broad spectrum metalloproteinase inhibitor inhibited the release of sIL-2Ralpha by PHA-activated peripheral blood mononuclear cells, suggesting that in contrast with current views concerning the major role of MMP-9 in the cleavage of membrane IL-2Ralpha, other proteases are involved in the shedding of sIL-2Ralpha. MMP-9 and sIL-2Ralpha appear therefore as independent prognostic markers in head and neck cancers.

A general algorithm for peak-tracking in multi-dimensional NMR experiments.

We present an algorithmic method allowing automatic tracking of NMR peaks in a series of spectra. It consists in a two phase analysis. The first phase is a local modeling of the peak displacement between two consecutive experiments using distance matrices. Then, from the coefficients of these matrices, a value graph containing the a priori set of possible paths used by these peaks is generated. On this set, the minimization under constraint of the target function by a heuristic approach provides a solution to the peak-tracking problem. This approach has been named GAPT, standing for General Algorithm for NMR Peak Tracking. It has been validated in numerous simulations resembling those encountered in NMR spectroscopy. We show the robustness and limits of the method for situations with many peak-picking errors, and presenting a high local density of peaks. It is then applied to the case of a temperature study of the NMR spectrum of the Lipid Transfer Protein (LTP).

Antioxidant capacities and phenolics levels of French wines from different varieties and vintages.

Phenolics from grapes and wines can play a role against oxidation and development of atherosclerosis. Levels of phenolics, major catechins [(+)-catechin, (-)-epicatechin, procyanidin dimers B1, B2, B3, and B4], phenolic acids (gallic acid and caffeic acid), caftaric acid, malvidin-3-glucoside, peonidin-3-glucoside, and cyanidin-3-glucoside were quantified by HPLC with UV detection for 54 French varietal commercial wines taken from southern France to study the antioxidant capacity and the daily dietary intake of these compounds for the French population. The highest antioxidant capacity was obtained with red wines and ranged from 12.8 mmol/L (Grenache) to 25.2 mmol/L (Pinot Noir). For white wines, Chardonnay enriched in phenolics by special wine-making was found to have an antioxidant capacity of 13.8 mmol/L, comparable to red wine values. For red wines classified by vintages (1996-1999) antioxidant capacities were approximately 20 mmol/L and then decreased to 13.4 mmol/L for vintages 1995-1991. Sweet white wines have 1.7 times more antioxidant capacity (3.2 mmol/L) than dry white wines (1.91 mmol/L). On the basis of a still significant French wine consumption of 180 mL/day/person, the current daily intake of catechins (monomers and dimers B1, B2, B3, and B4) averaged 5 (dry white wine), 4.36 (sweet white wines), 7.70 (rosé wines), 31.98 (red wines), and 66.94 (dry white wine enriched in phenolic) mg/day/resident for the French population. Red wine, and particularly Pinot Noir, Egiodola, Syrah, Cabernet Sauvignon, and Merlot varieties, or Chardonnay enriched in phenolics during wine-making for white varieties contribute to a very significant catechin dietary intake.

Acoustic scattering by a modified Werner method

A modified integral Werner method is used to calculate pressure scattered by an axisymmetric body immersed in a perfect and compressible fluid subject to a harmonic acoustic field. This integral representation is built as the sum of a potential of a simple layer and a potential of volume. It is equivalent to the exterior Helmholtz problem with Neumann boundary condition for all real wave numbers of the incident acoustic field. For elastic structure scattering problems, the modified Werner method is coupled with an elastodynamic integral formulation in order to account for the elastic contribution of the displacement field at the fluid/structure interface. The resulting system of integral equations is solved by the collocation method with a quadratic interpolation. The introduction of a weighting factor in the modified Werner method decreases the number of volume elements necessary for a good convergence of results. This approach becomes very competitive when it is compared with other integral methods that are valid for all wave numbers. A numerical comparison with an experiment on a tungsten carbide end-capped cylinder allows a glimpse of the interesting possibilities for using the coupling of the modified Werner method and the integral elastodynamic equation used in this research.

Critical role of human bisphosphoglycerate mutase Cys22 in the phosphatase activator-binding site.

The enzymatic activities catalyzed by bisphosphoglycerate mutase (BPGM, EC 5.4.2.4) have been shown to occur at a unique active site, with distinct binding sites for diphosphoglycerates and monophosphoglycerates. The physiological phosphatase activator (2-phosphoglycolate) binds to BPGM at an undetermined site. BPGM variants were constructed by site-directed mutagenesis of three amino acid residues in the active site to identify residues specifically involved in the binding of the monophosphoglycerates and 2-phosphoglycolate. Substitution of Cys22 by functionally conservative residues, Thr or Ser, caused a great decrease in 2-phosphoglycolate-stimulated phosphatase activity and in the Ka value of the activator, whereas it caused no change in other catalytic activities or in the Km values of 2,3-diphosphoglycerate (2,3-DPG) and glycerate 3-phosphate (3-PG, EC 1.1.1.12), indicating that Cys22 is specifically involved either directly or indirectly in 2-phosphoglycolate binding. Kinetic experiments showed that the Ka of the cofactor and the Km of 3-PG were affected by the substitution of Ser23 indicating that this residue is necessary for the fixation of both 3-PG and 2-phosphoglycolate. The R89K variant has previously been shown to have a modified Km value for monophosphoglycerates, however, its affinity for 2-phosphoglycolate is unaltered, suggesting that Arg89 is specifically involved in monophosphoglycerates binding. CD spectroscopic studies of substrates and cofactor binding showed that 2,3-DPG induced structural modifications of normal and mutated enzymes which could be due to protein phosphorylation. Addition of 2-phosphoglycolate to phosphorylated proteins with normal affinity for the cofactor produced spectra with the same characteristics as unphosphorylated species. In summary, monophosphoglycerates and 2-phosphoglycolate have partially distinct binding sites in human BPGM. The specific implication of the Cys22 residue in 2-phosphoglycolate binding is of great significance in the design of analogs of therapeutic benefit.

New procedures to measure synthase and phosphatase activities of bisphosphoglycerate mutase. Interest for development of therapeutic drugs.

In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentration is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bisphosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allow us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phosphoglycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM.

Affinity-labeling of an NADPH-binding site on the heavy subunit of flavocytochrome b558 in particulate NADPH oxidase from activated human neutrophils.

Cell stimulation of blood phagocytes activates the superoxide-producing NADPH oxidase. Cytochrome b558, one of the two oxidase redox components, comprises a light (alpha) and a heavy glycosylated (beta) subunit. The other redox component, a flavoprotein, is now thought to be the heavy subunit, on the basis of amino acid sequence comparisons and of reconstitution experiments with purified components. We published that pyridoxal-5'-diphospho-5'-adenosine is an inactivating affinity label for the NADPH-binding site of particulate oxidase from activated neutrophils. We have now radiolabeled the inactivated oxidase by reducing with Na[3H]BH4 the Schiff base formed between proteins and the reagent. Upon SDS-PAGE, the NADPH-inhibitable incorporation is found at the same position as the immunodetectable cytochrome heavy subunit, before and after deglycosylation. Membranes from either activated cells of a cytochrome-deficient X-linked granulomatous disease patient or normal resting cells show no incorporation at this position. Our results provide experimental evidence for the existence on the cytochrome b558 heavy chain of an NADPH-binding site which can only be affinity-labeled by PLP-AMP when the oxidase is active. This suggests the occurrence of a conformational change in the cofactor binding site upon enzyme activation.

Inactivation of NADPH oxidase from human neutrophils by affinity labeling with pyridoxal 5'-diphospho-5'-adenosine.

When a particulate NADPH oxidase prepared from phorbol ester-activated human neutrophils was treated with pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), the superoxide anion-producing activity was inhibited according to affinity labeling kinetics. NADPH afforded a protection against inactivation which was competitive with respect to PLP-AMP; 2',5'-ADP and 2'-phospho-5' diphosphoadenosine (ATP ribose) appeared to be as potent as NADPH as protecting agents. NADP+ and ATP were less effective, while ADP and GTP-gamma-S did not protect significantly. These results suggest that PLP-AMP can be used, in conjunction with tritiated cyanoborohydride, to identify the elusive NADPH-dependent flavoprotein which is part of the electron transfer chain of NADPH oxidase.

Polymeric analogues of N-formyl peptides are potent activators of degranulation and superoxide production by human neutrophils.

Analogues of N-formyl methionyl leucyl phenylalanine possessing three and four peptide units grafted onto an inert carbon skeleton were tested as activators of human polymorphonuclear leukocytes. For the two responses studied, degranulation and respiratory burst, the polymeric analogues showed two maxima of activity, one at the same concentration as the monomer, the other one at a concentration 100- to 1000-fold lower. The potency of the polymers with respect to the monomer is discussed in terms of receptor clustering. The similarity of the dose-response curves for superoxide production and lysozyme secretion indicates that the early transmembrane signalling events are identical for the two responses studied.