Comparative effectiveness of gonadotropins used for ovarian stimulation during assisted reproductive technologies (ART) in France: A real-world observational study from the French nationwide claims database (SNDS).

This comparative non-interventional study using data from the French National Health Database (Système National des Données de Santé) investigated real-world (cumulative) live birth outcomes following ovarian stimulation, leading to oocyte pickup with either originator recombinant human follicle-stimulating hormone (r-hFSH) products (alfa or beta), r-hFSH alfa biosimilars, or urinaries including mainly HP-hMG (menotropins), and marginally u-hFSH-HP (urofollitropin). Using data from 245,534 stimulations (153,600 women), biosimilars resulted in a 19% lower live birth (adjusted odds ratio (OR) 0.81, 95% confidence interval (CI) 0.76-0.86) and a 14% lower cumulative live birth (adjusted hazard ratio (HR) 0.86, 95% CI 0.82-0.89); and urinaries resulted in a 7% lower live birth (adjusted OR 0.93, 95% CI 0.90-0.96) and an 11% lower cumulative live birth (adjusted HR 0.89, 95% CI 0.87-0.91) versus originator r-hFSH alfa. Results were consistent across strata (age and ART strategy), sensitivity analysis using propensity score matching, and with r-hFSH alfa and beta as the reference group.

The development of an oral antidiabetic combination tablet: design, evaluation and clinical benefits for patients with type 2 diabetes.

Type 2 diabetes is a chronic and progressive disease. Oral antidiabetic monotherapies directly address only one defect as their primary mechanism of action, and do not control blood glucose sufficiently well to meet current glycaemic targets. In consequence, most patients need combination therapy within a few years. However, the co-administration of two or more oral antidiabetic drugs may render treatment regimens difficult to follow. Combining oral antidiabetic agents into a single tablet provides a means of intensifying antidiabetic therapy while supporting good patient compliance. An insulin sensitiser and an insulin secretagogue represent a rational oral antidiabetic combination, as they address the dual endocrine defects of insulin resistance and impaired beta-cell function in type 2 diabetes. Nevertheless, the components of a combination tablet must be carefully chosen. Metformin (an insulin sensitiser) and glibenclamide (an insulin secretagogue) are well supported by decades of clinical evidence, and the pharmacokinetics of these agents support twice-daily co-administration. The final technical challenge is to optimise their delivery within a single-tablet combination. A recently-introduced metformin-glibenclamide combination tablet (Glucovance) has been extensively studied in well-designed clinical trials, where it has been shown to be more effective than its component monotherapies in controlling fasting and postprandial glycaemia. This treatment provides a case study in the development of a single-tablet oral antidiabetic combination, in terms of the pharmacokinetic issues facing the development of this preparation, and the implications of the pharmacokinetic properties of the components of the combination tablet on their pharmacodynamic actions and risk-benefit profile.

Intracellular survival of Brucella spp. in human monocytes involves conventional uptake but special phagosomes.

Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated how B. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca(2+)- and Mg(2+)-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.

Brucella suis-impaired specific recognition of phagosomes by lysosomes due to phagosomal membrane modifications.

Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments.

Early events and implication of F-actin and annexin I associated structures in the phagocytic uptake of Brucella suis by the J-774A.1 murine cell line and human monocytes.

Brucella spp. are facultative, intracellular pathogenic bacteria that cause brucellosis, a zoonosis affecting mammalian species. Brucella entry into myelomonocytic cell lines is highly enhanced by opsonization. Few studies have been undertaken to unravel the first interactions between these bacteria and their host cells. This paper deals with early events following contact of Brucella suis with the J-774A.1 phagocytic cell line and differentiated monocytes. Phagocytic uptake of bacteria was documented under a fluorescence microscope using GFP-expressing B. suis. Unlike entry in the J-774A. 1 cell line, non-opsonized Brucella entered differentiated human monocytes as efficiently as opsonized bacteria. However, following 1 h infections, a mean of only three bacteria were phagocytized and the whole monocyte population was only infected after a 4 h infection. Contact of non-opsonized Brucella with phagocytes did not induce marked structural changes at the cell surface, as revealed by scanning electron microscopy. Contact of Brucella (opsonized or not) elicited transient local recruitment of F-actin, revealed by phalloidin labelling, and of annexin I-associated structures, revealed by immunofluorescence staining. Finally, bacteria appeared to be rapidly internalized in monocytes once they had adhered to the cell surface. A low percentage of infected cells and few adhered and/or internalized bacteria following short-term infections could have resulted either from the fact that there were few sites of entry or the weak bacterial initial interactions with the host-cell membrane or the bacterial receptor.

Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages.

Brucella suis is a facultative intracellular pathogen of mammals, residing in macrophage vacuoles. In this work, we studied the phagosomal environment of these bacteria in order to better understand the mechanisms allowing survival and multiplication of B. suis. Intraphagosomal pH in murine J774 cells was determined by measuring the fluorescence intensity of opsonized, carboxyfluorescein-rhodamine- and Oregon Green 488-rhodamine-labeled bacteria. Compartments containing live B. suis acidified to a pH of about 4.0 to 4.5 within 60 min. Acidification of B. suis-containing phagosomes in the early phase of infection was abolished by treatment of host cells with 100 nM bafilomycin A(1), a specific inhibitor of vacuolar proton-ATPases. This neutralization at 1 h postinfection resulted in a 2- to 34-fold reduction of opsonized and nonopsonized viable intracellular bacteria at 4 and 6 h postinfection, respectively. Ammonium chloride and monensin, other pH-neutralizing reagents, led to comparable loss of intracellular viability. Addition of ammonium chloride at 7 h after the beginning of infection, however, did not affect intracellular multiplication of B. suis, in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h. Thus, we conclude that phagosomes with B. suis acidify rapidly after infection, and that this early acidification is essential for replication of the bacteria within the macrophage.

[Molecular dynamics of annexin I in normal and infected cells]. / Dynamique moléculaire de l'annexine I dans la cellule normale et infectée.

Annexins are a family of proteins present within the tissues of all multicellular organisms, mammalian erythrocytes excepted. The property shared by all annexins is the calcium and membrane binding. Annexins are constituted of two domains. The N-terminal domain gives the molecule its specificity and the C-terminal domain, highly conserved, containing 4 repetitions of 70 amino-acids, gives the common properties. Although numerous important works were performed, the exact function of annexins is not unraveled. They participate to many cellular processes as for instance exocytosis, endocytosis or phagosome maturation. Many hypotheses, supported by experimental results, have been proposed. In this review, we propose a summary of the principal characteristics of annexins and we discuss the main hypotheses proposed for their functions.

Structural analysis of junctions formed between lipid membranes and several annexins by cryo-electron microscopy.

The (annexin II-p11)2 tetramer has been proposed to participate in exocytosis and several other members of the annexin superfamily have been reported to aggregate liposomes in vitro. In this context, the Ca2+-dependent binding of several annexins to chromaffin granules and liposomes was investigated by cryo-electron microscopy. The Ca2+-dependent aggregation of lipid membranes by (annexin II-p11)2 results from the spontaneous self-organization of the protein into two-dimensional plaques, which are visualized in projection as characteristic junctions. The junctions have a constant thickness of 210(+/-10) A and present a symmetrical distribution of electron-dense material arranged into seven stripes. They were observed over a wide range of Ca2+ concentrations, down to 2 microM. The molecular components corresponding to the seven electron-dense stripes were assigned as follows: the two associated membranes give rise to two outer stripes each and the three central stripes correspond to the (annexin II-p11)2 tetramer. Each annexin II molecule interacts with the outer lipid leaflet of one membrane, giving rise to one stripe, while the central stripe is due to the (p11)2 dimer with which both annexin II molecules interact. Both annexin II and annexin I also induced the Ca2+-dependent aggregation of liposomes via junctions that lack the central (p11)2 moiety and present only six high-density stripes. As expected, both annexin V and annexin III bind to liposomes without inducing their aggregation.

Effects of profilin-annexin I association on some properties of both profilin and annexin I: modification of the inhibitory activity of profilin on actin polymerization and inhibition of the self-association of annexin I and its interactions with liposomes.

We have previously shown that annexin I, a member of a family of calcium-dependent phospholipid and membrane binding proteins, interacts with profilin with high specificity and affinity. This finding further suggests that annexin I is involved through profilin in the regulation of membrane-cytoskeleton organization. We have investigated the consequences of a complex formed by these two proteins on the functions of both profilin and annexin I. Annexin I is able to modify the inhibitory effect of profilin on actin polymerization. This action is partial and the mechanism involved appears to be complex. On the other hand, the association between annexin I and profilin is sufficiently strong to inhibit the self-association of annexin I. The binding capacity of annexin I to liposomes containing phosphatidylserine, which mimics annexin I binding to membranes, is also decreased by profilin. This binding is nevertheless restored when phosphatidylinositol 4,5-biphosphate (PtdInsP2) is included in the liposomes. Finally, the capacity of annexin I to aggregate liposomes is also modified. It is worthwhile mentioning that the liposomes-binding and liposomes-aggregating activities of annexin I are independently regulated. The cell localization and functions of annexin I and profilin suggest that interaction between these two proteins may be directly implicated in the regulation of membrane-cytoskeleton. The phospholipid composition of membranes may be one of the modulating factors.

Characterization of the interaction between annexin I and profilin.

Annexin I belongs to a family of calcium-dependent phospholipid-binding and membrane-binding proteins. Although many of the biochemical properties and the three-dimensional structure of this protein are known, its true physiological roles have yet to be thoroughly defined. Its putative functions include participation in the regulation of actin microfilaments dynamics, proposed after the discovery of an interaction with actin. In accordance with this hypothesis, we found that annexin I can also interact with profilin. We used different methods, overlay and surface plasmon resonance (BIAcore), to measure the parameters of the association equilibrium, i.e. k(on), k(off) and k(d). The affinity of annexin I for profilin was between 10(7) M and 10(8) M. High concentrations of KCl did not prevent the interaction, although a slight decrease in affinity was observed. Calcium, a modulator of annexin I functions interfered only marginally with the association, in a manner comparable to magnesium. Proteins or compounds known to interact with annexin I or profilin were found to inhibit the annexin-I--profilin interaction when added in the reaction medium. Recombinant profilin exhibited a slightly lower affinity than natural platelet protein when measured with BIAcore. Due to the submembrane localisation of annexin I and the regulatory activity of profilin on the cytoskeleton, an interaction between annexin I and profilin may therefore be implicated in the regulation of some cellular functions, particularly those governing membrane-cytoskeleton dynamic organization.

End-tidal carbon dioxide during cardiopulmonary resuscitation in humans presenting mostly with asystole: a predictor of outcome.

OBJECTIVE: To determine whether continuous semiquantitative assessment of end-tidal CO2 could provide a highly sensitive predictor of return of spontaneous circulation during cardiopulmonary resuscitation (CPR). DESIGN: Prospective, clinical study. SETTING: Prehospital CPR. PATIENTS: One hundred twenty patients, during nontraumatic cardiac arrest. INTERVENTIONS: End-tidal CO2 values were measured continuously after tracheal intubation, and were categorized as the initial value, and as minimal and maximal values during the first 20 mins. MEASUREMENTS AND MAIN RESULTS: Presenting rhythm was asystole in 22 of the first 24 patients. Return of spontaneous circulation occurred in eight patients. Initial, minimal, and maximal end-tidal CO2 values were significantly (p < .01) higher in these patients than in the patients without return of spontaneous circulation. Cutoff values providing a 100% sensitivity and the highest specificity in predicting return of spontaneous circulation were found to be 10 torr for initial and maximal end-tidal CO2 values, and 2 torr for the minimal end-tidal CO2 value. The number of patients required to reject (with a risk error of <.05) the hypothesis of an actual sensitivity of < or = 90% for an observed sensitivity of 100% was found to be 95. In the second part of the study, this hypothesis was prospectively tested for initial and maximal end-tidal CO2 values in the subsequent 96 patients. Presenting cardiac rhythm was asystole in 87 patients. Return of spontaneous circulation was obtained in 30 patients. The cutoff value of 10 torr for maximal end-tidal CO2 during the first 20 mins after tracheal intubation provided an observed sensitivity of 100% in predicting return of spontaneous circulation with a specificity of 67%. This result allows rejection of the hypothesis of an actual sensitivity of < or = 90% (p = .042). By contrast, the observed sensitivity of initial end-tidal CO2 was only 87%. CONCLUSIONS: End-tidal CO2 represents a valuable tool for monitoring patients presenting with asystole during prehospital CPR. Fluctuations in end-tidal CO2 during CPR and the utility of end-tidal CO2 in detecting return of spontaneous circulation justify its continuous measurement. In addition, a high sensitivity (>90%) in predicting return of spontaneous circulation is prospectively demonstrated using the maximal end-tidal CO2 during the first 20 mins after tracheal intubation, with a cutoff value of 10 torr. Such a prognostic indicator could be used for a more rational approach to prolonged CPR.

Change in the N-terminal domain conformation of annexin I that correlates with liposome aggregation is impaired by Ser-27 to Glu mutation that mimics phosphorylation.

Annexin I is a member of the annexin family of calcium-dependent membrane binding proteins. The core domain of these proteins is similar in all annexins but the N-terminal domain is specific for each member. This domain is thought to regulate annexin function through phosphorylation. In annexin I, Ser-27 is one of the amino acids that can be phosphorylated by protein kinase C. Phosphorylations are thought to regulate some annexin I functions by increasing calcium requirement. Two mutants were prepared in this study: S27E and S27A proteins. The first mimics phosphorylation while the second prevents phosphorylation at residue 27. Wild-type annexin I and S27A mutant protein showed the same calcium dependence for phospholipid vesicles aggregation, while S27E mutant protein showed a higher calcium requirement and a low maximal extent of aggregation. By contrast, liposome binding and self-association required identical calcium concentrations for the wild-type and the two mutant proteins. To examine whether the regulation observed is due to modification of the N-terminal structure, we investigated conformational changes by using two approaches. Firstly we analysed proteinase sensibility. Limited proteolysis of the N-terminal tail showed similar patterns for the three proteins. Using drastic conditions of proteolysis, we observed strong resistance of the core domain to digestion in the presence of calcium. Secondly, since Ser-27 is located on the N-terminal domain that contains a tryptophan located at position 12, the fluorescence of this residue was analysed during Ca2+-binding of wild-type annexin I and S27E mutant protein. The results demonstrated that Ca2+ induces a slight change in the Trp environment of wild-type annexin I, corresponding to a burying of the residue. No changes in fluorescence features were observed with S27E mutant protein. The results obtained show that phosphorylation of the N-terminal tail plays a regulatory role in phospholipid vesicle aggregation, which is based on a mechanism distinct from protein self-association. This phosphorylation induces a conformational change in the tail probably related to aggregation property.

Distribution of annexin I during non-pathogen or pathogen phagocytosis by confocal imaging and immunogold electron microscopy.

Annexin I is an abundant protein in U937 cells differentiated towards a macrophagic phenotype. These cells become able to kill Escherichia coli, however, the intracellular pathogen Brucella suis, known to interfere with phagosome maturation, multiply in these differentiated cells. We have analysed by confocal and electron microscopy the cellular localization of annexin I during phagocytosis of yeast, non-pathogenic E. coli and the intracellular pathogen B. suis. Using immunocytochemical detections annexin I was found mainly as patches in the cytoplasm of uninfected cells. Upon phagocytosis of yeast or E. coli organisms, annexin I rapidly translocated and concentrated around phagosomes. On the other hand, annexin I was never detected around live B. suis-containing phagosomes. However, when dead brucellae were used, annexin I did translocate to the periphagosomal region. Our results suggest that annexin I could play a role in the molecular mechanism of phagosome maturation, which is impaired by some intracellular pathogens.

Neuromuscular effects of rocuronium on the diaphragm and adductor pollicis muscles in anesthetized patients.

BACKGROUND: Rocuronium has properties that may make it suitable for rapid-sequence intubation. However, its neuromuscular effects have been studied only on the adductor pollicis. This study compares the neuromuscular effect of rocuronium on the diaphragm and adductor pollicis in humans. METHODS: The forces generated by the diaphragm and the adductor pollicis during supramaximal single-twitch stimulation of the phrenic and ulnar nerves, respectively, were studied during thiopental, fentanyl, and nitrous oxide-oxygen anesthesia. In 6 patients, cumulative doses of 0.15, 0.25, 0.35, 0.45, and 0.60 mg.kg-1 rocuronium were given over a 9-min period. The doses for 50% (ED50) and 95% (ED95) depression of twitch height were calculated. In another 12 patients, the times for maximal effect and 10%, 25%, 50%, 75%, and 90% recovery of the twitch height were calculated after a bolus dose of 0.60 mg.kg-1 rocuronium. RESULTS: ED50 and ED95 were higher for the diaphragm (0.26 +/- 0.07 and 0.50 +/- 0.20 mg.kg-1, respectively) than for the adductor pollicis (0.14 +/- 0.05 and 0.24 +/- 0.04 mg.kg-1). Rocuronium 0.60 mg.kg-1 produced 100% paralysis of the adductor pollicis in all patients and of the diaphragm in 9 of 12 patients. The onset time for muscle relaxation after 0.6 mg.kg-1 rocuronium was shorter for the adductor pollicis than for the diaphragm (80 +/- 20 vs. 120 +/- 62 s). Times for 10%, 25%, 75%, and 90% recovery of twitch height were 34 +/- 10, 40 +/- 13, 56 +/- 20, and 64 +/- 21 min, respectively, for the adductor pollicis, and significantly shorter for the diaphragm: 17 +/- 10, 23 +/- 9, 33 +/- 13, and 35 +/- 10 min, respectively. CONCLUSIONS: The diaphragm is more resistant than the adductor pollicis to rocuronium, as shown by greater ED50 and ED95 and faster recovery of the twitch height. The intubating dose of 0.60 mg.kg-1 is close to the ED95 of 0.50 mg.kg-1 for the diaphragm.

[Truncal blocks of the lower limb]. / Blocs tronculaires du membre inférieur.

Plexus nerve blocks of the lower limb have been described for many years but were seldom used until recently. Postoperative analgesia is one of the main indications of these blocks. The blockade of both lumbar and sciatic plexuses is required for most of the surgical procedures performed on the proximal part of the lower limb. Nerve stimulators account for less difficulties to perform such blocks and results are more reliable. Several pharmacokinetic studies have documented that toxic thresholds of plasma concentrations of local anaesthetics are not reached with the doses commonly injected. Many different techniques and landmarks have been described providing several alternatives to perform these blocks according to the type and the localisation of the surgical procedure. Extensive indications are reported for day case surgery or patients at risk.

[Plexus nerve blocks for postoperative analgesia after orthopedic surgery of the lower limbs]. / Blocs nerveux tronculaires à visée analgésique après chirurgie orthopédique des membres inférieurs.

In our institution, plexus nerves blocks have been performed in seventy patients after lower limb surgery. The technique is considered as simple and reliable. The administration of a 0.375 per cent bupivacaine epinephrine containing solution allows to obtain analgesia longer than 15 hours in 45 p. 100 of the patients, devoided of side effects. Lower limb plexus nerves blocks appear as an efficient technique for postoperative analgesia following orthopaedic surgery.

[Analysis of failures of spinal anesthesia as a function of practice development in a university hospital]. / Analyse des échecs de la rachianesthésie en fonction de l'évolution de la pratique d'un hôpital universitaire.

This study is a retrospective analysis of 303 consecutive spinal anaesthesia performed in orthopaedic patients of a University Hospital between January and December 1990. Failure of spinal anaesthesia was defined as the requirement for general anaesthesia to perform surgery. The parameters studied as possible risk factors of failure were patients demographics, local anaesthetic agents and solutions and techniques of spinal anaesthesia (single injection versus continuous spinal anaesthesia). Failures were related to inadequate or incomplete extension of sensory blockade or to difficulties to perform spinal injection. Continuous spinal anaesthesia was performed in 209 patients mostly with 0.5% isobaric bupivacaine, while 94 patients received a single injection of either hyperbaric 0.5% tetracaine with adrenaline or 0.5% bupivacaine or 5% lidocaine. Failures occurred in 6.3% of the cases but were significantly less frequent with continuous spinal anaesthesia (4.8%) than with the conventional technique (9.6%). The incidence of failure was higher with hyperbaric tetracaine (11.1%) confirming its poor reliability. Inadequate extension of the anaesthetic block was the main cause of failure whatever the spinal anaesthetic technique. These results point out the reliability of continuous spinal anaesthesia but problems may occasionally occur due to spinal catheter misplacement.

Interactions of plasma gelsolin with actin. Isolation and characterization of binary and ternary plasma-gelsolin-actin complexes.

We have studied the interactions between plasma gelsolin and actin: firstly the complex formation between both proteins, secondly the effects of gelsolin and its complexes on G-actin polymerization and F-actin fragmentation. Complex formation has been studied by high-performance gel permeation chromatography; plasma gelsolin alone elutes at an Mr of about 77000 and a Stokes radius of 3.7 nm; complex formation occurs in the presence of Ca2+: by chromatography in the presence of EGTA, a binary complex is obtained with an Mr of 134000 and a Stokes radius of 4.7 nm; and by chromatography in the presence of Ca2+, a ternary complex is obtained with an Mr of 173000 and a Stokes radius of 5.2 nm. The binary complex is EGTA-stable. In relation to this stability of the binary complex, the depolymerizing function of gelsolin is not reversed upon chelation of Ca2+. The effects of plasma gelsolin and its complexes on both G-actin polymerization and F-actin fragmentation, and their Ca2+ dependence have been examined by viscometry and electron microscopy. The main conclusions of these studies are the following: the fast processes are the formation of ternary complex, which acts as a heteronucleus for G-actin polymerization, and the severing function of gelsolin, these fast processes are Ca2+-dependent; the slow processes are related to the capping ability of gelsolin or its complexes and are Ca2+-independent.