Novel immunomic technologies for schistosome vaccine development.

Schistosomiasis remains one of the most common human helminthiases, despite the availability of an effective drug against the causative parasites. Drug treatment programmes have several limitations, and it is likely that a vaccine is required for effective control. While decades of vaccine development have seen the discovery and testing of several candidate antigens, none have shown consistent and acceptable high levels of protection. The migrating larval stages are susceptible to immunity, however few larval-specific antigens have been discovered. Therefore, there is a need to identify novel larval-specific antigens, which may prove to be more efficacious than existing targets. Immunomics, a relatively new field developed to cope with the recent large influx of biological information, holds promise for the discovery of vaccine targets, and this review highlights some immunomic approaches to schistosome vaccine development. Firstly, a method to focus on the immune response elicited by the important and vulnerable larval stage is described, which allows a targeted study of the immunome at different tissue sites. Then, two high-throughput arrays are discussed for the identification of protein and carbohydrate antigens. It is anticipated that these approaches will progress vaccine development against the schistosomes, as well as other parasites.

[Conservative treatment of endometrial cancer and atypical hyperplasia]. / Traitement conservateur du cancer et des hyperplasies atypiques de l'endomètre.

Endometrial carcinoma is the most frequent pelvic cancer encountered in women. The discovery of an endometrial carcinoma in a woman seeking pregnancy cannot be considered as exceptional. The medical alternative to the classic radical surgical treatment is studied in a review. Treatment with progestins might be considered and discussed with the couple in special indications. The oncologic risk to which this treatment exposes is limited. However, the application and the surveillance of this therapeutic protocol must obey strict rules, in order to recognize without delay any resistance to treatment. The spontaneous fertility of such patients seems weak, most probably because of their age, but assisted reproductive techniques (ART) could be considered in particular cases. Secondary hysterectomy is controversial, but a recent tendency is to widen this practice is becoming apparent.

[Systematic hysteroscopy prior to in vitro fertilization]. / Hystéroscopie systématique avant fécondation in vitro.

OBJECTIVE: To evaluate the benefits of a diagnostic hysteroscopy prior to in vitro fertilization. PATIENTS AND METHODS: We retrospectively studied 145 patients who underwent ICSI during a period of 6 months. Office hysteroscopy was systematically performed before the first stimulation cycle. If pathological findings were revealed, appropriate medical or surgical treatment was given. RESULTS: Pathological patterns were observed in 45% of hysteroscopies. Endometritis, polyps and myomas and mucosal diseases were the most frequently observed. The patients aged over 38 years didn't show higher rate of pathology (29% vs 27% for the younger patients). The treatment of pathologies gave the same pregnancy rate than the normal cavities. Patients with endometritis were treated with antibiotics and 40% of them became pregnant in the following cycle. DISCUSSION AND CONCLUSION: Systematic hysteroscopy prior to IVF-ICSI showed to be an effective investigation that could improve the pregnancy rate.

[Degradation of copper IUDs in utero. The process of metallic corrosion. A scanning electron microscope study]. / Dégradation in utero des DIU au cuivre en fonction du temps. Le phénomène de corrosion métallique. Etude au microscope électronique à balayage.

OBJECTIVE: The present investigation was carried out in order to study the process of metallic corrosion of copper IUD's in utero, to precise its dynamics and location along the IUD and to appraise the influence of eventual calcareous deposition. MATERIAL AND METHODS: A total of 461 copper IUDs representing four standard models were screened by means of optical microscopy. Especially typical samples were studied at higher magnifications under the scanning electron microscope. The obtained data were considered in terms of statistics. RESULTS: It was possible to demonstrate a preferential corrosive activity in the lower part of the IUD without significant variations between the models. It was also possible to precise the steps of the process, to describe its most characteristic aspects and to study the eventual effect of severe calcareous deposition on corroded copper. DISCUSSION AND CONCLUSIONS: Intrauterine copper corrosion is a normal process which occurs preferentially in the cervical portion of an IUD and can lead to the total metal loss. Both its initiation and evolution are submitted to strong individual variations. Thick and compact vaterite deposits may thwart copper erosion in case of drastic and rapid deposition.

The structure of synthetic oligosaccharides in relation to factor IXa inhibition.

We investigated the effect of various oligosaccharides (OS) on the inhibition of factor IXa by antithrombin (AT) in a purified system. The OS comprised the AT-binding pentasaccharide sequence prolonged by saccharide chains with various lengths and charges. We show that factor IXa inhibition depended on the molecular weight of the OS. Factor IXa was not inhibited by the AT-binding pentasaccharide alone, but was inhibited if it was prolonged by a sulphated dodecasaccharide at the non-reducing end. The overall charge was also important since factor IXa inhibition was negligible if the pentasaccharide was prolonged by a non-sulphated dodecasaccharide. Using compounds containing a non-sulphated spacer, we showed that the central part of the OS was not critical. This study therefore demonstrates that the minimal OS structure necessary for catalysing factor IXa inhibition by AT is close to that required for catalysing thrombin inhibition.

[I.U.D. degradation in utero as a function of time: the phenomena of calcification. Radiocrystallographic and scanning electron microscopy study]. / Dégradation des D.I.U. in utero en fonction du temps: le phénomène de calcification. Etude radiocristallographique et au microscope électronique à balayage.

OBJECTIVE: A study was undertaken in order to determine the frequency of the process of calcareous deposition, to clarify the nature of the deposits, their arrangement along the IUD and their possible influence on the appearance of metrorrhagias. MATERIAL AND METHODS: A total of 408 coopper IUD's belonging to four standard models were observed by means of optical microscopy at low magnifications. The most typical samples were studied under the scanning electron microscope and deposits analyzed by crystallography. The data were analysed statistically. RESULTS: It was possible to determine both the frequency and the importance of the process of calcification according to the model of IUD and to the bearing time, to demonstrate a preferential location of concretions and to suspect a sensibility to the phenomenon variable according to the model. It was also possible to clarify the stages of the process of calcification, to describe its most characteristic aspects and to identify the essential component of calcareous deposits. CONCLUSIONS: Calcification concerns roughly over 50% of IUD's in utero. It occurs preferentially on the fundic portion of an IUD. Obviously, the minimal time required for the phenomenon to start as well as to increase its intensity are submitted to strong individual variations. Concretions are composed of vaterite, a distinct variety of carbonate of calcium. They do not seem to be the primary cause of possible metrorrhagias.

SR123781A, a synthetic heparin mimetic.

SR123781A, a synthetic hexadecasaccharide comprising an antithrombin (AT) binding domain, a thrombin binding domain, and a neutral methylated hexasaccharide sequence, was obtained from glucose through a convergent synthesis. SR123781A showed high affinity for human AT (Kd = 58 +/- 22 nM) and was a potent catalyst of its inhibitory effect with regard to factor Xa (IC50) = 77 +/- 5 ng/ml - 297 +/- 13 U/mg) and thrombin (IC50 = 4.0 +/- 0.5 ng/ml - 150 +/- 30 U/mg). SR123781A which acted exclusively via AT (no effect via heparin cofactor II at a concentration of 6 microg/ml) inhibited thrombin generation occurring via both the extrinsic and intrinsic pathways in vitro in human plasma. SR123781A did not compete for 3H-heparin binding to PF4 and did not activate platelets in the presence of plasma from patients with heparin-induced thrombocytopenia. After intravenous or subcutaneous administration to rats, rabbits or baboons, SR123781A displayed prolonged anti-factor Xa and anti-factor IIa activity ex vivo. After intravenous injection to baboons, decreases of the anti-factor Xa and anti-thrombin activities were parallel and disappeared with the same pharmacodynamics. Intravenous administrations of SR123781A strongly inhibited thrombus formation in an experimental model of thromboplastin-induced venous thrombosis in rats with an ED50 value of 18 +/- 0.1 microg/kg (vs 77 +/- 3 microg/kg for heparin). SR123781A inhibited arterial thrombus formation induced on a silk thread in an arterio-venous shunt and in the vena cava (ED50 values of 225 +/- 10 and 27 +/- 8 microg/kg, respectively). Compared to standard and low molecular weight heparin and to presently used drugs, SR123781A exhibited a highly favourable antithrombotic/bleeding ratio therefore showing that it might be considered as a promising compound in the treatment and prevention of various thrombotic diseases.

Experimental proof for the structure of a thrombin-inhibiting heparin molecule.

Kinetic studies of thrombin inhibition by antithrombin in the presence of heparin have shown that thrombin binds to heparin in a preformed heparin-antithrombin complex. To study the relative position of the thrombin binding domain and the antithrombin binding domain on a heparin molecule we have designed and synthesized heparin mimetics, which structurally are very similar to the genuine polysaccharide. Their inhibitory properties with respect to factor Xa and thrombin provide experimental evidence that in heparin the thrombin binding domain must be located at the nonreducing end of the antithrombin binding domain to observe thrombin inhibition. As expected, factor Xa inhibition is not affected by elongation of the antithrombin binding pentasaccharide sequence, regardless of the position in which this elongation takes place.

Synthesis of conformationally locked L-iduronic acid derivatives: direct evidence for a critical role of the skew-boat 2S0 conformer in the activation of antithrombin by heparin.

We have used organic synthesis to understand the role of L-iduronic acid conformational flexibility in the activation of antithrombin by heparin. Among known synthetic analogues of the genuine pentasaccharidic sequence representing the antithrombin binding site of heparin, we have selected as a reference compound the methylated anti-factor Xa pentasaccharide 1. As in the genuine original fragment, the single L-iduronic acid moiety of this molecule exists in water solution as an equilibrium between three conformers 1C4, 4C1 and 2S0. We have thus synthesized three analogues of 1, in which the L-iduronic acid unit is locked in one of these three fixed conformations. A covalent two atom bridge between carbon atoms two and five of L-iduronic acid was first introduced to lock the pseudorotational itinerary of the pyranoid ring around the 2S0 form. A key compound to achieve this connection was the D-glucose derivative 5 in which the H-5 hydrogen atom has been replaced by a vinyl group, which is a progenitor of the carboxylic acid. Selective manipulations of this molecule resulted in the 2S0-type pentasaccharide 23. Starting from the D-glucose derivative 28, a covalent two atom bridge was now built up between carbon atoms three and five to lock the L-iduronic acid moiety around the 1C4 chair form conformation, and the 1C4-type pentasaccharide 43 was synthesized. Finally the L-iduronic acid containing disaccharide 58 which, due to the presence of the methoxymethyl substituent at position five adopts a 4C1 conformation, was directly used to synthesize the 4C1-type pentasaccharide 61. The locked pentasaccharide 23 showed about the same activity as the reference compound 1 in an antithrombin-mediated anti-Xa assay, whereas the two pentasaccharides 43 and 61 displayed very low activity. These results clearly establish the critical importance of the 2S0 conformation of L-iduronic acid in the activation of antithrombin by heparin.

Identification of a hexasaccharide sequence able to inhibit thrombin and suitable for 'polymerisation'.

Three hexasaccharides, having from low to very high affinity for antithrombin, were synthesised from disaccharide building block precursors. One of them, methyl(sodium 2,3-di-O-methyl-4-O- sodium sulfonato-alpha-L-idopyranosyluronate)-(1-->4)-[(2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranosyl)-(1-->4)-(sodium 2,3-di-O-methyl-alpha-L-idopyranosyluronate)-(1-->4)]2-2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranoside, obtainable from a single disaccharide building block precursor, constitutes a good starting point for obtaining simple oligosaccharidic heparin mimetics able to inhibit the two coagulation factors thrombin and Factor Xa.

Assessment through chemical synthesis of the size of the heparin sequence involved in thrombin inhibition.

Deca- to eicosasaccharides having the generic structure methyl(sodium 2,3-di-O-methyl-4-O-sodium sulfonato-alpha-L-idopyranosyluronate)-(1-->4)-[(2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranosyl)-(1-->4)-(sodium 2,3-di-O-methyl-alpha-L-idopyranosyluronate)-(1-->4)]n-2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranoside have been synthesized from a single disaccharide precursor. All of them bind to and activate antithrombin. When n < or = 6 only Factor Xa inhibition is observed, whereas when n > 6 Factor Xa and thrombin are both inhibited in the presence of antithrombin. These results indicate that, in heparin, the sequence involved in antithrombin-catalyzed thrombin inhibition is a pentadeca- or a hexadecasaccharide.

[New antithrombotic oligosaccharides]. / Nouveaux oligosaccharides antithrombotiques.

In the early eighties, following breakthroughs in oligosaccharide chemistry, the total chemical synthesis of pentasaccharides has been achieved, representing the antithrombin binding domain of heparin (the active site). The selective inhibitors of coagulation factor Xa thus obtained represent a new type of antithrombotic drugs. In a further step, based on the knowledge of the mechanism of antithrombin activation by heparin, oligosaccharides (pentadeca- to eicosasaccharides), comprising an antithrombin binding domain prolonged by a thrombin binding domain, were designed and synthesised in the Sanofi group. These compounds inhibit both factor Xa and thrombin, in the presence of antithrombin. Endowed with the full anticoagulant activity of heparin but devoid of undesired non-specific interactions, particularly with platelet factor 4 (PF4), they might represent "the ideal heparin-like antithrombotic".

New synthetic heparin mimetics able to inhibit thrombin and factor Xa.

Synthetic pentadeca-, heptadeca- and nonadecasaccharides, comprising an antithrombin III (AT III) binding pentasaccharide prolonged at the non-reducing end by a thrombin binding domain have been obtained. The pentadecasaccharide is the shortest oligosaccharide able to catalyse thrombin inhibition by AT III. The nonadecasaccharide is a more potent thrombin inhibitor than standard heparin.

Synthetic oligosaccharides having various functional domains: potent and potentially safe heparin mimetics.

A synthetic heptadecasaccharide, comprising an antithrombin III binding domain, a thrombin binding domain, and a neutral methylated hexasaccharide sequence, was obtained through a convergent synthesis. This compound displayed in vitro anticoagulant properties similar to that of standard heparin but, in contrast with heparin, escaped neutralization by platelet factor 4, a protein released by activated platelets.

Synthesis of thrombin-inhibiting heparin mimetics without side effects.

Unwanted side effects of pharmacologically active compounds can usually be eliminated by structural modifications. But the complex heterogeneous structure of the polysaccharide heparin has limited this approach to fragmentation, leading to slightly better-tolerated heparin preparations of low molecular mass. Despite this improvement, heparin-induced thrombocytopaenia (HIT), related to an interaction with platelet factor 4 (PF4) and, to a lesser extent, haemorrhages, remain significant side effects of heparinotherapy. Breakthroughs in oligosaccharide chemistry made possible the total synthesis of the pentasaccharide antithrombin-binding site of heparin. This pentasaccharide represents a new family of potential antithrombotic drugs, devoid of thrombin inhibitory properties, and free of undesired interactions with blood and vessel components. To obtain more potent and well-tolerated antithrombotic drugs, we wished to synthesize heparin mimetics able to inhibit thrombin, that is, longer oligosaccharides. Like thrombin inhibition, undesired interactions are directly correlated to the charge and the size of the molecules, so we had to design structures that were able to discriminate between thrombin and other proteins, particularly PF4. Here we describe the use of multistep converging synthesis to obtain sulphated oligosaccharides that meet these requirements.

[Atomic absorption spectrophotometry study of copper ion release by copper-bearing intrauterine devices]. / Etude par spectrométrie d'absorption atomique du largage des ions cuivre (Cu++) par les dispositifs intra-utérins au cuivre.

Copper release from copper-bearing IUD's was studied in vitro and in vivo using atomic absorption spectrophotometry in deionized water, normal saline solution and normal ovulatory cervical mucus. In these media, copper release from a 375 mm2 DIU occurs without latency, showing comparable amounts for identical time intervals. Daily copper release was shown to be respectively 8 and 11 times higher in cervical mucus and normal saline solution than in deionized water. Although copper ions are detectable in ovulatory cervical mucus under physiological conditions, the copper content appears 5 to 6 times higher in women bearing a copper IUD. Obviously, the copper amount is dependent on the copper exposed surface: the daily in vitro release from a 250 mm2 IUD is 18% inferior to that observed from a 375 mm2 model. In vivo, the daily copper release in ovulatory mucus of 380 or 200 mm2 IUD users is respectively 5 and 3.5 times higher than in controls.

A novel synthetic reversible inhibitor of sialidase efficiently blocks secondary but not primary influenza virus infection of MDCK cells in culture.

The sodium salts of 2-difluoromethyl-phenyl-alpha-ketoside of N-acetyl-neuraminic acid (compound 1) and of 4-difluoromethyl-2-methoxy-phenyl-alpha-ketoside of N-acetylneuraminic acid (compound 2) were designed as potential mechanism-based inhibitors of sialidase. In vitro both of these compounds competitively inhibited the sialidases of Clostridium perfringens and of influenza virus A/HK/1/68. Inhibition was irreversible with the sialidase of Clostridium perfringens whereas it was reversible with that of A/HK/1/68. Compound 2 did not inhibit the hemagglutinin of the virus but exhibited significant anti-influenza activity when added to the medium of Madin-Darby canine kidney (MDCK) cells infected by influenza virus. In non-infected MDCK cells no inhibition of cellular sialidase was observed. Compound 2 did not block primary infection, but inhibited the release of progeny virus from infected cells. Even after 8 passages in its presence, no resistant strains were detected. Because of its high Ki (8 x 10(-5) M) compared to the low Ki (1' x 1(-10) M) of 4 guanidino-Neu 5 Ac 2en and its reversible inhibition of viral sialidase, its development as an anti-influenza agent is no longer envisaged. Nevertheless, as a mechanism-based irreversible inhibitor of the bacterial enzyme, it could at least be useful for investigating the intrinsic role of sialidase in infections caused by this strain.

A novel synthetic reversible inhibitor of sialidase efficiently blocks secondary but not primary influenza virus infection of MDCK cells in culture.

The sodium salts of 2-difluoromethyl-phenyl-α-ketoside of N-acetyl-neuraminic acid (compound 1) and of 4-difluoromethyl-2-methoxy-phenyl-α-ketoside of N-acetylneuraminic acid (compound 2) were designed as potential mechanism-based inhibitors of sialidase. In vitro both of these compounds competitively inhibited the sialidases of Clostridium perfringens and of influenza virus A/HK/1/68. Inhibition was irreversible with the sialidase of Clostridium perfringens whereas it was reversible with that of A/HK/1/68. Compound 2 did not inhibit the hemagglutinin of the virus but exhibited significant anti-influenza activity when added to the medium of Madin-Darby canine kidney (MDCK) cells infected by influenza virus. In non-infected MDCK cells no inhibition of cellular sialidase was observed. Compound 2 did not block primary infection, but inhibited the release of progeny virus from infected cells. Even after 8 passages in its presence, no resistant strains were detected. Because of its high Ki (8 × 10-5M) compared to the low Ki (1 × 1-10 M) of 4 guanidino-Neu 5 Ac 2en and its reversible inhibition of viral sialidase, its development as an anti-influenza agent is no longer envisaged. Nevertheless, as a mechanism-based irreversible inhibitor of the bacterial enzyme, it could at least be useful for investigating the intrinsic role of sialidase in infections caused by this strain.