Kinetic analysis and binding studies of a new recombinant human factor VIIa for treatment of haemophilia.

INTRODUCTION/AIM: LR769 is a new second-generation recombinant human Factor VIIa (rhFVIIa) developed for haemophilia treatment. We determined enzymatic properties of LR769 and its interaction with antithrombin, tissue factor, platelets and endothelial protein C receptor (EPCR), compared with NovoSevenRT. METHODS: Kinetic enzyme assays and active site titration were used for enzymatic studies. Surface Plasmon Resonance (SPR) was used for determination of binding constants. Cellular binding was determined for platelets and cultured human umbilical vein endothelial cells (HUVEC). RESULTS: The dissociation constant (Kd ) for activated platelet binding was in the 1 µm range for both products. At saturation, more LR769 than NovoSevenRT was bound to the platelets. Binding to HUVEC was 25-50% higher for LR769 than for NovoSevenRT. Protein C, soluble EPCR, and anti-EPCR antibody all reduced the binding, indicating specificity for EPCR. LR769 was similar to NovoSevenRT in all kinetic assays. Active site titration demonstrated 0.7 mole of active site/mole of protein. The kcat /Km values for activation of FX and FIX with purified recombinant tissue factor and phospholipids were 10.5 s-1 /0.32 µm and 3.3 s-1 /0.44 µm respectively. The apparent second-order rate constant for inactivation by human plasma AT was 5.9 ± 0.4 × 103 m-1 s-1 . The Kd values for binding of LR769 to soluble tissue factor and full-length tissue factor were 8.1 nm and 0.9 nm, respectively, and the Kd for binding to soluble EPCR was 41 nm. CONCLUSION: Overall, LR769 exhibited characteristics similar to NovoSevenRT, but bound EPCR on HUVEC with somewhat higher affinity than NovoSevenRT.

3D-shape recognition and size measurement of irregular rough particles using multi-views interferometric out-of-focus imaging.

We realize simplified-tomography experiments on irregular rough particles using interferometric out-of-focus imaging. Using two angles of view, we determine the global 3D-shape, the dimensions, and the 3D-orientation of irregular rough particles whose morphologies belong to families such as sticks, plates, and crosses.

Dual-wavelength digital holography for 3D particle image velocimetry: experimental validation.

A multi-exposure digital in-line hologram of a particle field is recorded by two successive pulses of different wavelengths. During the reconstruction step, each recording can be independently analyzed by selecting a given wavelength. This procedure enables avoiding the superimposition of particle images that may be close to each other.

Simultaneous 3D location and size measurement of bubbles and sand particles in a flow using interferometric particle imaging.

We present a system to characterize a triphasic flow in a 3D volume (air bubbles and solid irregular particles in water) using only one CCD sensor. A cylindrical interferometric out-of-focus imaging setup is used to determine simultaneously the 3D position and the size of bubbles and irregular sand particles in a flow. The 3D position of the particles is deduced from the ellipticity of their out-of-focus image. The size of bubbles is deduced from analysis of interference fringes. The characteristics of irregular sand particles are obtained from analysis of their speckle-like pattern. Experiments are confirmed by simulations.

In vitro and in vivo properties differ among liquid intravenous immunoglobulin preparations.

OBJECTIVE: To compare in vitro and in vivo biological and biochemical properties of five liquid intravenous immunoglobulin (IVIg) preparations licensed for therapeutic use in Europe. METHODS: ClairYg(®) was compared in a blinded manner to four other liquid IVIg preparations licensed in Europe (Octagam(®) , Kiovig(®) , Gamunex(®) , Privigen(®) ). Three batches of each preparation were tested, except for the IgG repertoires and the animal model. RESULTS: Levels of anti-A and anti-B antibodies were lower in ClairYg(®) (0·11/0·11) relative to a positive EDQM standard and Octagam(®) (0·11/0·08) than in other preparations (0·33-0·69/0·42-0·46). IgG in ClairYg(®) recognized 365 and 416 protein spots in HEp-2 cell and Escherichia coli protein extracts vs. 230-330 and 402-842 protein spots, respectively, for IgG in other preparations. IgA content (301 vs. 165-820 ng/mg of IgG), Factor XI and Factor XII antigen (0·46 vs. 0·85-2·40 mU/mg of IgG and 7·8 vs. 20·0-46·2 lU/mg of IgG) C1q binding (0·42 vs. 0·67-1·89 arbitrary units) and C5a uptake (0·41 vs. 0·45-0·66% of activation) were lower in ClairYg(®) than in other preparations. Finally, intravenous infusion of ClairYg(®) , Gamunex(®) and Privigen(®) had no major effect on arterial blood pressure in spontaneously hypertensive rats. CONCLUSIONS: Our results evidence some differences in the biological and biochemical properties among licensed liquid IVIg preparations.

Metabolomic profiling with NMR discriminates between biphosphonate and doxorubicin effects on B16 melanoma cells.

The metabolomic profiles of B16 melanoma cells were investigated in vitro with high resolution-magic angle spinning proton magnetic resonance spectroscopy and OPLS multivariate statistical analyse. We compared the profiles for untreated melanoma B16-F10 cells and Ca(2+) chelating EGTA, doxorubicin or BP7033 bisphosphonate treated cells. The two last molecules are known to induce anti-proliferative effects by different mechanisms of action in cells. Untreated and EGTA treated cells had similar profiles and were considered together as control cells. Several spectral regions could discriminate control from doxorubicin as well as BP7033 treated cells. Doxorubicin and BP7033 displayed distinct metabolic profiles. Important changes in neutral lipids and inositol were related to doxorubicin activity whereas BP7033 affected essentially phospholipids and alanine/lactate metabolism. These results provide new putative targets for both drugs. Metabolomics by NMR is shown here to be a good tool for the investigation of the mechanisms of action of drugs in pre-clinical studies.

Pharmacological strategies and micrometastasis: what is known? What must be done?

Metastasis accounts for over 90% of cancer patient deaths. In spite of this , the majority of the currently used drugs are aimed at eradicating or controlling the primary tumor. Hardly any cancer therapies used at the moment interfere only with metastasis and there is a need for drugs acting specifically on the metastatic processes, curing the minimal residual disease and being less toxic than the current drugs. Searching for drugs that do not affect the growth of the primary tumor but inhibit the colonization and/or the outgrowth in distant sites could lead to new drugs specifically aimed at treating the minimal residual disease. The aim of this work is to search at what conditions such drugs might be discovered and if some exist already. For that, mechanisms of actions of current experimental and clinical antimetastatic drugs are described and their utility for different clinical settings (primary prevention, adjuvant setting) is analyzed. In conclusion, clues exist that specific antimetastatic drugs could exist and will be developed in a near future.

Aptamer-Based Affinity Chromatography for Protein Extraction and Purification.

Aptamers are oligonucleotide molecules able to recognize very specifically proteins. Among the possible applications, aptamers have been used for affinity chromatography with effective results and advantages over most advanced protein separation technologies. This chapter first discusses the context of the affinity chromatography with aptamer ligands. With the adaptation of SELEX, the chemical modifications of aptamers to comply with the covalent coupling and the separation process are then extensively presented. A focus is then made about the most important applications for protein separation with real-life examples and the comparison with immunoaffinity chromatography. In spite of well-advanced demonstrations and the extraordinary potential developments, a significant optimization work is still due to deserve large-scale applications with all necessary validations. Graphical Abstract Aptamer-protein complexes by X-ray crystallography.

Microsatellite instability and sensitivitiy to FOLFOX treatment in metastatic colorectal cancer.

BACKGROUND: Microsatelite instability (MSI) is the consequence of the inactivation of a mismatch repair gene and is observed in approximately 15% of colon cancer cases. Patients with MSI colon cancer do not benefit from 5-fluorouracil (5-FU)-based chemotherapy. A current treatment of reference for colon cancer is a combination of 5-FU and oxaliplatin (FOLFOX). The aim of this study was to determine the efficiency of the FOLFOX treatment in patients with metastatic MSI colon cancer. PATIENTS AND METHODS: Tumour specimens were collected from patients with metastatic colon cancer treated with FOLFOX 4 modified or FOLFOX 6; these two regimens are based on 85 mg/m2 and 100 mg/m2 oxaliplatin, respectively. The MSI status was assessed by measuring the length of five monomorphic mononucleotide markers. The FOLFOX regimen was evaluated as a first-line treatment according to WHO criteria. RESULTS: Forty patients (22 men, 18 women), median age 63.5 years (27-83 years) were treated with FOLFOX 4 or 6. Nine patients had tumours exhibiting high MSI (MSI group) and 31 patients had tumours exhibiting microsatellite stability (MSS group). In the MSS group, 11 partial responses (36%) were observed, while there were only two in the MSI group (22%) (no significant difference). The two patients who were responders in the MSI group were treated with FOLFOX 6. The overall survival was not significantly different for MSI and MSS patients. CONCLUSION: No significant differences in the overall response rate or overall survival between the two groups of patients were observed. However, these results suggest that patients with MSI colon cancer are more sensitive to a higher dose of FOLFOX.

Direct measurement of the mechanism by which magnesium specifically modifies the mechanical properties of DNA.

We examine the effect of physiological cations Na+, K+, Mg2+, and Ca2+ on the mechanical properties of bundles of λ-phage DNA using silicon nanotweezers (SNTs). Integrating SNTs with a microfluidic device allows us to perform titration experiments while measuring the effect in real-time. The results show that only for Mg2+ and in particular, at the intra-nuclear concentration (100 mM), the interaction occurs.

Sudden, unexpected death due to undiagnosed frontal glioblastoma in a schizophrenic patient.

The incidence of sudden death due to undiagnosed primary intracranial tumor is low in forensic autopsy. We report a case of a 48-year-old white male, known to be a schizophrenic patient for several years, and in whom a medico-legal autopsy disclosed a large, previously undiagnosed, bilateral frontal glioblastoma infiltrating the genu of corpus callosum. We emphasize the importance of performing complete autopsy, including a thorough neuropathological examination, in all cases of sudden unexpected death, especially in those cases in which no extracerebral cause of death had been established and whose clinical history was primarily of a psychiatric nature.

[About a case of uterine per-partum rupture, 37months after resection of a rectovaginal endometriosis nodule]. / À propos d'un cas de rupture utérine per-partum, 37 mois après une résection d'un nodule recto-vaginal d'endométriose.

Endometriosis is a common condition in women, whose main repercussions are painful symptoms. In addition, it was shown that endometriosis was a major cause of infertility and various obstetric complications could be related to this pathology. Uterine rupture is a rare but serious complication whose incidence tends to decrease with the screening of women at risk, however, its fetal, maternal morbidity and mortality causes remains important. We were confronted with a case of posterior uterine rupture in a patient of 36 years, primipare term exceeded in immediate postpartum period. The patient's primary antecedent of uterine surgery torus was responsible for infertility endometriosis. The outcome was favorable for the mother, after a surgical treatment by laparotomy, and for the child. In the literature, two cases have been reported of uterine rupture after endometriosis surgery, which is why we found it interesting to report this rare case. Given the increase in surgical management of this disease, it seems relevant to ask whether, in the future, we should be more vigilant in monitoring pregnancy for these women.

Integrative analysis of gene expression patterns predicts specific modulations of defined cell functions by estrogen and tamoxifen in MCF7 breast cancer cells.

To explore the mechanisms whereby estrogen and antiestrogen (tamoxifen (TAM)) can regulate breast cancer cell growth, we investigated gene expression changes in MCF7 cells treated with 17beta-estradiol (E2) and/or with 4-OH-TAM. The patterns of differential expression were determined by the ValiGen Gene IDentification (VGID) process, a subtractive hybridization approach combined with microarray validation screening. Their possible biologic consequences were evaluated by integrative data analysis. Over 1000 cDNA inserts were isolated and subsequently cloned, sequenced and analyzed against nucleotide and protein databases (NT/NR/EST) with BLAST software. We revealed that E2 induced differential expression of 279 known and 28 unknown sequences, whereas TAM affected the expression of 286 known and 14 unknown sequences. Integrative data analysis singled out a set of 32 differentially expressed genes apparently involved in broad cellular mechanisms. The presence of E2 modulated the expression patterns of 23 genes involved in anchors and junction remodeling; extracellular matrix (ECM) degradation; cell cycle progression, including G1/S check point and S-phase regulation; and synthesis of genotoxic metabolites. In tumor cells, these four mechanisms are associated with the acquisition of a motile and invasive phenotype. TAM partly reversed the E2-induced differential expression patterns and consequently restored most of the biologic functions deregulated by E2, except the mechanisms associated with cell cycle progression. Furthermore, we found that TAM affects the expression of nine additional genes associated with cytoskeletal remodeling, DNA repair, active estrogen receptor formation and growth factor synthesis, and mitogenic pathways. These modulatory effects of E2 and TAM upon the gene expression patterns identified here could explain some of the mechanisms associated with the acquisition of a more aggressive phenotype by breast cancer cells, such as E2-independent growth and TAM resistance.

[Prediction of spontaneous preterm birth in symptomatic patients: A review]. / Prédiction de la prématurité en cas de menace d'accouchement prématuré: revue de la littérature.

OBJECTIVE OF THE REVIEW: To identify predictors of preterm delivery in the context of threatened preterm labour. MAIN POINTS: Tobacco use and previous history of preterm delivery are the main anamnestic elements to predict preterm birth. High positive predictive value of vaginal examination is restricted to cases with strong cervical alterations like dilatation over 4 cm. In case of discrete cervical alterations, literature confirms the great interest for cervical length ultrasonographic measurement as it reduces false positive cases. Absence of fetal respiratory movements appears to be as sensitive as cervical length and could be more specific but its clinical use remains rare. Vaginal detection of fetal fibronectin is the most useful biomarker with high negative predictive value (>90%). Fibronectin quantitative test seems to enhance the positive predictive value. No other biomarker is currently used in clinical practice. Electromyography and elastography of the cervix appear to be promising approaches.

Effects of aerosolized pentamidine on glucose homeostasis and insulin secretion in HIV-positive patients: a controlled study.

OBJECTIVE: Intravenous pentamidine induces hypo- and hyperglycaemia (dose-dependent toxicity on islet beta cells), pancreatitis and nephrotoxicity. Conversely, aerosolized pentamidine (AP) is usually devoid of systemic side-effects: few reports of hypo- or hyperglycaemia have been published. Our study aimed to assess the influence on glucose homeostasis and insulin secretion of long-term exposure to AP used for prophylaxis of Pneumocystis carinii pneumonia in HIV-positive patients, and to compare the impact on insulin secretion of AP, whether administered for the first time or after prolonged monthly exposure. DESIGN: Retrospective cross-sectional controlled study (main objective) and non-randomized prospective controlled study. PATIENTS: We compared glucose homeostasis and C peptide response to 1 mg intravenous glucagon in patients who had previously inhaled > or = 10 prophylactic aerosols (group 1, n = 21) and in HIV-positive controls (groups 2 and 3, n = 28) who had received none. Both groups were comparable for age and body-mass index, but CD4 T-lymphocyte counts and Karnofsky scores were both significantly higher in the control group. RESULTS: Fasting (T0) blood glucose, fructosamine and response to the first glucagon test were similar in both groups, but postprandial glucose, glycated haemoglobin and fasting C peptide were significantly higher (P < 0.05) in the pentamidine group. A second glucagon test was performed on the same day, 3 h (T3) after AP inhalation in 35 patients (in 21 after > or = 10 aerosols, group 1; in 14 after the first, group 2) and in 14 HIV-positive controls (group 3). The only significant difference between the three groups in C peptide response to this second test was a lower peak T3/peak T0 ratio in group 1. Plasma amylase and creatinine were not altered by the aerosol. CONCLUSION: Long-term prophylactic exposure to AP had minor but significant effects on glucose homeostasis and insulin secretion but did not modify pancreatic and renal function. The detrimental effects induced by long-term exposure to AP found in our study are probably not clinically relevant, but a more prolonged exposure to AP might conceivably induce more severe alterations.

Evidence of direct thyroid-stimulating action of thyrotropin-releasing hormone by perifusion of rat thyroid fragments.

The possibility that TRH has a direct thyroid-stimulating action has never been reported. A number of studies have shown a rise in serum concentrations of thyroid hormone after stimulation with TRH, without a rise in TSH secretion. This has led us to test TRH with rat thyroid fragment perifusion. Significant T4 release was observed for TRH concentrations as low as 1.7 X 10(-11) M. A dose-response curve was determined. The response was immediate, reaching a peak after the sixth minute and continuing for 15 min after the stimulation had ceased. This kinetic pattern is different from the one observed with TSH and with theophylline and suggests that a different mechanism may be involved. TRH seems to be capable of directly stimulating both the secretion of the pituitary hormone (TSH) and the corresponding peripheral gland, like LHRH, which also acts directly on the Leydig cells of the testis.

Effects on bone mass of long term treatment with thyroid hormones: a meta-analysis.

Osteoporosis is the main cause of spine and hip fractures. Morbidity, mortality, and costs arising from hip fractures have been well documented. Thyroid hormones (TH) are widely prescribed, mainly in the elderly. Some studies (but not all) found a deleterious effect of suppressive TH therapy on bone mass. These conflicting data raised a controversy as to the safety of current prescribing and follow-up habits, which, in turn, raised major health-care issues. To look for a detrimental effect on bone of TH therapy, we performed a meta-analysis (by pooling standardized differences, using a fixed effect model) of all published controlled cross-sectional studies (41, including about 1250 patients) concerning the impact of TH therapy on bone mineral density (BMD). Studies with women receiving estrogen therapy were excluded a priori, as were studies with a high percentage of patients with postoperative hypoparathyroidism, when no separate data were available. We decided to stratify the data according to anatomical site, menopausal status, and suppressive or replacement TH therapy, resulting in 25 meta-analysis on 138 homogeneous subsets of data. The main sources of heterogensity between studies that we could identify were replacement or suppressive TH therapy, menopausal status, site (lumbar spine, femoral neck, Ward's triangle, greater trochanter, midshaft and distal radius, with various percentages of cortical bone), and history of hyperthyroidism, which has recently been found to impair bone mass in a large epidemiological survey. To improve homogeneity, we excluded a posteriori 102 patients from 3 studies, who had a past history of hyperthyroidism and separate BMD data, thus allowing assessment of the TH effect in almost all 25 subset meta-analyses. However, controls were usually not matched with cases for many factors influencing bone mass, such as body weight, age at menarche and at menopause, calcium dietary intake, smoking habits, alcohol intake, exercise, etc. For lumbar spine and hip (as for all other sites), suppressive TH therapy was associated with significant bone loss in postmenopausal women (but not in premenopausal women), whereas, conversely, replacement therapy was associated with bone loss in premenopausal women (spine and hip), but not in postmenopausal women. The detrimental effect of TH appeared more marked on cortical bone than on trabecular bone. Only a large long term prospective placebo-controlled trial of TH therapy (e.g. in benign nodules) evaluating BMD (and ideally fracture rate) would provide further insight into these issues.

Arachnoid cysts in children.

Twenty-seven cases of intracranial arachnoid cysts in children aged 1 to 16 years have been studied with attention to diagnosis, pathologic findings, and surgical treatment. In nine patients, the cyst was located in the posterior fossa. In eight patients, there were other CNS or other organ system abnormalities. No specific symptom complex can be ascribed to arachnoid cysts in that the clinical manifestations are the result of their size and location, secondary complications, and remote effects. This is illustrated in the ten cases reported.

Decrease of carbohydrate in membrane glycoproteins during human erythrocyte ageing in vivo.

Membrane glycoproteins from young human erythrocytes and erythrocytes aged in vivo were fractionated by gel filtration. Three major groups of glycoproteins were obtained. The neutral hexoses and sialic acid contents of each group of glycoproteins from the old cells were found to be significantly reduced by comparison to the values found in the glycoproteins of the young cells. Thus, the previously observed decrease in neutral hexoses and in sialic acid contents of the full erythrocyte membrane during in vivo ageing does not affect only one particular group of glycoproteins but each of the groups of glycoproteins tested, including the major glycoproteins of the erythrocyte membrane, that is to say glycophorins. It is shown in addition, that the previously observed decrease per cell of the surface galactose and N-acetylgalactosamine residues of the ageing erythrocyte affect several groups of membrane glycoproteins including band 3, PAS-1, PAS-4 and PAS-3. The physiological significance of these experimental data is discussed.

The role of T-agglutinin in the disappearance of erythrocytes artificially aged by desialylation.

The chance discovery of two mouse strains, one with and one without a high presence of serum T-agglutinin, permitted the investigation of the role of this antibody in the disappearance of desialylated erythrocytes, which may be regarded as a model for ageing. The proportional relationship between the quantity of sialic acid removed and the diminution of half-life is not affected by the presence or absence of T-agglutinin. Opsonization by T-agglutinin would therefore appear to be an improbable mechanism. Other possible theories are discussed.