A synthetic kisspeptin analog that triggers ovulation and advances puberty.

The neuropeptide kisspeptin and its receptor, KiSS1R, govern the reproductive timeline of mammals by triggering puberty onset and promoting ovulation by stimulating gonadotrophin-releasing hormone (GnRH) secretion. To overcome the drawback of kisspeptin short half-life we designed kisspeptin analogs combining original modifications, triazole peptidomimetic and albumin binding motif, to reduce proteolytic degradation and to slow down renal clearance, respectively. These analogs showed improved in vitro potency and dramatically enhanced pharmacodynamics. When injected intramuscularly into ewes (15 nmol/ewe) primed with a progestogen, the best analog (compound 6, C6) induced synchronized ovulations in both breeding and non-breeding seasons. Ovulations were fertile as demonstrated by the delivery of lambs at term. C6 was also fully active in both female and male mice but was completely inactive in KiSS1R KO mice. Electrophysiological recordings of GnRH neurons from brain slices of GnRH-GFP mice indicated that C6 exerted a direct excitatory action on GnRH neurons. Finally, in prepubertal female mice daily injections (0.3 nmol/mouse) for five days significantly advanced puberty. C6 ability to trigger ovulation and advance puberty demonstrates that kisspeptin analogs may find application in the management of livestock reproduction and opens new possibilities for the treatment of reproductive disorders in humans.

[Results of a novel real-time PCR, sequence analysis, Inno-LiPA line probe assays in the detection of hepatitis B virus G1896A precore mutation in French blood donors]. / Résultats de trois méthodes pour la détection de la mutation précore G1896A du virus de l'hépatite B chez les donneurs de sang français : PCR temps réel, séquençage et test Inno-LIPA.

AIM: To screen hepatitis B virus (HBV) genotypes and associated basal core promoter (BCP; T1762A/A1764) and precore (PC; A1896) mutations among the 100 HBV surface antigen (HBsAg) positive voluntary blood donors in France. METHODS: HBV genotypes were determined by using direct sequence analysis. Three methods were used to detect G1896A mutation: non-commercial real-time PCR (PCRTR°, line probe assay (InnoLiPA HBV PreCore, INNOGENETICS(®)) and direct sequencing of precore gene. HBV viral load was quantified with two commercial real-time PCR (COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV Test/Roche and Real Time HBV/M2000/Abbott). RESULTS: The mean age of donors was 30 (18-64). Patients were from Africa (42%), Europa (50%), and Asia (8%). HBV/D was the most predominant (37%) genotype followed by HBV/A (31%) and HBV/E (22%). PC and BCP mutants were found in 57% with Inno-LIPA HBV test and 59% with both PCRTR and sequencing methods. A significant difference in the viral load of blood donors with wild and PC mutants was observed with the Taqman Cobas real time PCR (3,19 Log(10) UI/ml versus 4,93 Log(10) UI/ml, p < 0.05). Precore phenotype determination was in agreement with the three PC mutation detection methods in 56% of cases. CONCLUSIONS: Non-Caucasian genotype E was present in the French blood donors. PC mutation was more common than BCP mutations in this study. As HBV infected blood donors were more often asymptomatic carriers, we could speculate that the G1896A mutation may favour the asymptomatic state, supporting previous observations.

Quantitative and qualitative control of cytotoxic preparations by HPLC-UV in a centralized parenteral preparations unit.

The constantly growing incidence of cancer and long-term treatment are leading to an increasing number of cytotoxic preparations in hospital pharmacies. Security and quality standards of cytotoxic preparations are essential to assure treatment efficiency and limit iatrogenic toxicity. In order to secure the process of cytotoxic preparations; we decided to install a quantitative and qualitative High Performance Liquid Chromatography (HPLC) control of cytotoxic preparations carried inside our pharmacotechnic unit. A 100 microl sample of each preparation was assayed by HPLC with ultraviolet/visible-diode array detection, which enabled the identification of all cytotoxic agents thanks to their characteristic UV spectra. We developed rapid and specific HPLC assays that determined qualitatively and quantitatively the presence of 21 different cytotoxic agents in less than 3.5 min. A fifteen per cent tolerance from the theoretical concentration was chosen in agreement with preparation and dosage bias, and a first period control of more than 4400 preparations revealed that around 7.7% preparations did not conform. The main objective of these controls was to avoid the administration of defective chemotherapies to patients and finally to use their results to identify error factors; as a result we will take corrective measures in order to reduce error frequency.

[Assessment of dermatological emergencies in a French university hospital]. / Evaluation d'une consultation d'urgences en dermatologie.

INTRODUCTION: The aim of our study was to understand the motivations of outpatients who come to dermatological emergencies in a university hospital. PATIENTS AND METHOD: This 6-week prospective study included outpatients who came to the dermatology emergency unit. This consultation is proposed each morning (from 8 to 9), from Mondays to Fridays. A questionnaire was distributed to outpatients. They answered questions on the functioning of this consultation and their own symptoms. The consulting dermatologist answered questions on the referring physician, the really urgent characteristics of the disease and the diagnosis. RESULTS: Patients were satisfied by the functioning of the consultation. Indeed, 59 p. 100 of outpatients thought that the timetable was convenient and 70 p. 100 that the delay before getting a consultation was rapid. 75 p. 100 felt they needed treatment rapidly. Nonetheless, 45 p. 100 did not think they had a serious disease. More than half of the outpatients were referred by their general practitioner; the others came spontaneously, or were referred by other departments or general emergencies. The most frequent diagnoses were cutaneous infections (27.6 p. 100), eczema (21 p. 100), then benign tumors, psoriasis, physical dermatoses, viral eruptions... DISCUSSION: A consultation for dermatological emergencies appears to reply to patients' demands. Nonetheless, most of these outpatients do not present with real dermatological emergencies. Criteria for real emergencies needs to be further defined and understood by citizens.

The Perseus Exobiology mission on MIR: behaviour of amino acids and peptides in Earth orbit.

Leucine, alpha-methyl leucine and two peptides were exposed to space conditions on board the MIR station during the Perseus-Exobiology mission. This long duration space mission was aimed at testing the delivery of prebiotic building blocks. During this mission, two amino acids (leucine and alpha-methyl leucine) and two peptides (leucine-diketopiperazine and trileucine thioethylester) were exposed in Earth orbit for three months. Basalt, clay and meteorite powder were also mixed with the samples in order to simulate the effects of potential meteorite protection. Analysis of the material after the flight did not reveal any racemization or polymerisation but did provide information regarding photochemical pathways for the degradation of leucine and of the tripeptide. Amino acids appeared to be more sensitive to UV radiation than peptides, the cyclic dipeptide being found to be as particularly resistant. Meteorite powder which exhibits the highest absorption in Vacuum UltraViolet (VUV) afforded the best protection to the organic molecules whereas montmorillonite clay, almost transparent in VUV, was the least efficient. By varying the thickness of the meteorite, we found that the threshold for efficient protection against radiation was about 5 microm. The possible exogenous origin of biological building blocks is discussed with respect to the stability to the molecules and the nature of the associated minerals.

In-source fragmentation of peptide aldehydes and acetals: influence of peptide length and charge state.

The use of in-source collision-induced dissociation (CID) was evaluated to generate structural information on peptide aldehydes, which represent an important class of compounds as inhibitors for serine and cysteine proteases and as key intermediates for protein engineering. By studying five peptide aldehydes of different lengths, and their peptide acetal counterparts, mass to charge (m/z) dependency of in-source fragmentation was established for peptides that differ only by their C-terminal functionalization. In-source fragmentation of peptide aldehydes and acetals leads to the same final ion, probably via a similar mechanism. Moreover, the gas-phase information obtained here reflects the equilibrium occurring in solution between the peptide aldehyde and its hydrated form, which was retained during the ionization process. The equilibrium constant was determined to be close to unity. Disturbance of this equilibrium should enable the stability of covalent hydration of a given series of aldehydes to be compared.

Synthesis and fluorescence properties of Oregon Green 514 labeled peptides.

Oregon Green 514, with high photostability and a pKa of 4.8, is suitable for imaging applications. Labeling of peptide-bound resin with the carboxylic acid form was optimized. Peptide conjugation resulted in altered fluorescence properties of the dye including a quenching of the intensity compared with that in aqueous buffers with a slight red-shift of the emission maximum.

Structural features of a chimeric peptide inducing cytotoxic T lymphocyte responses in saline.

Little information is available correlating the structural properties of peptides with their immunogenicity in terms of responses via cytotoxic T lymphocytes (CTLs). The TT-NP6 chimeric peptide, consisting of two copies of a promiscuous T-helper epitope (T: residues 288-302 from the fusion protein of the measles virus) linked to the NP6 T-cytotoxic epitope (NP6: residues 52-60 from the nucleoprotein of measles virus) was able to induce virus-specific CTL responses in the absence of any adjuvant and hydrophobic component. The present work was undertaken to gain insight into structural features of the TT-NP6 peptide that may be important in optimizing the CTL immunogenicity of the peptide. Circular dichroism data, obtained in a buffer of physiological ionic strength and pH, strongly suggest a self-associated state for the peptide, which was confirmed by a sedimentation velocity experiment. However, helix association is accompanied by loss of overall helical content. Thermal-dependence studies show that the unfolding of self-associated alpha-helices is significantly more pronounced than the unfolding of isolated alpha-helices. Circular dichroism data, together with tryptic limited proteolysis, suggest the presence of a charged amino acid within the hydrophobic core. This study should provide a basis for engineering more effective immunogenic peptides against the measles virus by increasing the stability of the TT-NP6 peptide.

Fine specificity of the antibody response to a synthetic peptide from the fusion protein and protection against measles virus-induced encephalitis in a mouse model.

A synthetic peptide representing residues 397-420 from the measles virus (MV) fusion (F) protein was tested for its structure, immunogenicity and protective capacity against intracerebral challenge with a neuroadapted strain of MV. Analysis of the peptide by mass spectrometry showed that it was linear, despite the presence of two cysteine residues in the sequence. Circular dichroism spectroscopy highlighted a weak preference for the peptide to adopt an alpha-helical conformation. The peptide was shown to be immunogenic in BALB/c and C57BL/6 mice after intraperitoneal immunization in Freund's adjuvant, and anti-peptide antibodies from both strains of mice reacted with the MV as a solid phase antigen on an ELISA plate. When the fine specificity of the anti-peptide antibody response was examined using overlapping 8-mer peptides, serum antibodies from BALB/c mice recognized the region between residues 407-417 whereas antibodies from C57BL/6 mice recognized the region 408-420 of the 397-420 peptide sequence. Although anti-397-420 antibodies had no demonstrable neutralizing activity, protection against challenge with a neuroadapted strain of MV was demonstrated following active immunization with peptide in C57BL/6 mice or after passive transfer of anti-peptide antibodies in BALB/c mice. These findings highlight the importance of the 397-420 region in the induction of protective antibodies in the MV encephalitis mouse model, and suggest that this epitope might be a good candidate for inclusion in a future MV synthetic peptide vaccine.

Structural properties of chimeric peptides containing a T-cell epitope linked to a fusion peptide and their importance for in vivo induction of cytotoxic T-cell responses.

We have previously shown that when administered to mice without adjuvant, a chimeric peptide consisting of the fusion peptide F from measles virus protein linked at the C-terminus of a cytotoxic T-cell epitope from the M2 protein of respiratory syncytial virus efficiently primes for an major histocompatibility complex (MHC) class-I restricted cytotoxic T lymphocyte (CTL) response. In this report, we demonstrated by microspectrofluorometry that the fusion-peptide moiety bound to the plasma membrane of living cells. When the fusion peptide was linked to the C-terminus of the CTL epitope, the chimeric peptide (M2-F) adopted a marked beta-sheet conformation. In contrast, when the fusion peptide was linked to the N-terminus of the T-cell epitope (F-M2), the chimeric peptide adopted an alpha-helical conformation in the presence of trifluoroethanol. The immunogenicity of the two chimeric peptides for class-I restricted CTL was also significantly different, the one adopting the alpha-helical conformation being more immunogenic. Probably due to its obvious conversion to an alpha-helical conformation, the F-M2 peptide could have a higher propensity to insert into membranes, as shown by microspectrofluorometry, with a resultant better immunogenicity than the M2-F peptide.

Structural requirements for synthetic immunogens to induce measles virus specific CTL responses.

We have studied the immunogenicity of a synthetic peptide representing a cytotoxic T cell epitope (CTL) from the nucleoprotein of measles virus (MV). For the induction of peptide and MV-specific CTL responses after subcutaneous immunization, covalent linkage of the CTL epitope to a T-helper epitope was required. The presence of two copies of the T-helper epitope at the amino terminus of the CTL epitope (TT-CTL) resulted in the induction of strong CTL responses after administration in saline. In contrast, a chimeric peptide with one copy of the T-helper epitope at the amino terminus of the CTL epitope (T-CTL) was weakly immunogenic when given in saline. Analysis of the structure of the TT-CTL chimeric peptide by CD spectroscopy revealed an alpha-helical conformation, as compared to the random coil conformation favored by the T-CTL chimeric peptide. In addition, the CD spectra of the TT-CTL peptide in the presence of small unilamellar vesicules (SUV) revealed an increased helicity, as compared to the spectra of the T-CTL chimera in the presence of SUV. This suggests that the amphipathic character of the TT-CTL chimeric construct favors its interaction with the cell membrane of antigen presenting cells, therefore, facilitating its cytosolic delivery for class I presentation. These findings highlight the importance of antigen structure for the in vivo induction of CTL responses and may have implications for the design of synthetic peptide vaccines.

High thermal stability and cold-denaturation of an artificial polypeptide.

An amphipathic polypeptide, Pn, with a tandemly repeated LKELPEKL sequence including a proline every eight residues, as well as a series of shorter peptides having the same sequence, P2, P3, P4, P5 and P6, were synthesized. Their conformation in aqueous solution was mainly studied by CD. At low temperature, these peptides and polypeptides are completely unordered and undergo a reversible transition leading to a partly alpha-helical structure upon heating. Such behavior has been demonstrated for a few proteins by other authors and has been called cold-denaturation. The transition temperature of the polypeptide is close to 20 degrees C. The conformational change does not depend on concentration, indicating a monomolecular process. The high-temperature structure seems to be compact as for globular proteins. A model of folded structure is proposed from experimental data and from molecular modelling studies.

Linkage of a fusion peptide to a CTL epitope from the nucleoprotein of measles virus enables incorporation into ISCOMs and induction of CTL responses following intranasal immunization.

The introduction of soluble protein antigens into the endogenous processing pathway is a prerequisite for the efficient induction of MHC class-1 restricted cytotoxic T-lymphocytes (CTLs). Antigens incorporated into immunostimulating complexes (ISCOMs) containing lipids and Quil-A are able to induce CD8+ CTL responses in vivo. Furthermore, lipopeptides have also been used to raise peptide-specific CTLs and bypass the requirement for the use of an adjuvant. Although conventional ISCOM technology is in general restricted to the use of hydrophobic proteins or fatty acid-derivitized proteins or peptides, we have demonstrated that the linkage of a conserved paramyxovirus fusion peptide to a CTL epitope NP29 (residues 281-290 of measles virus nucleoprotein) resulted in the incorporation of this hydrophilic CTL epitope into ISCOMs and the in vivo priming of peptide specific CTLs following intranasal immunization. In addition, the fusion peptide-CTL epitope chimera was able to efficiently sensitise P815 target cells for lysis by nucleoprotein specific CTLs induced following immunization of mice with recombinant RAd-68 adenovirus, suggesting the efficient introduction of the peptide into the class-1 restricted antigen processing pathway. Furthermore, immunization of mice with this fusion peptide chimera in saline was able to prime an NP29-specific CTL response despite the absence of adjuvant.

The binding of chimeric peptides to GM1 ganglioside enables induction of antibody responses after intranasal immunization.

With a model peptide, the neutralizing epitope 50-75 of cholera toxin B subunit, two chimeric peptides were constructed. A T-cell epitope, the 174-187 peptide from the G protein of the respiratory syncytial virus, was co-linearly synthesized at the amino-(174-50) or carboxyl-terminus (50-174) of the 50-75 peptide. Although both chimeric peptides were equally immunogenic by the intraperitoneal route, the 50-174 peptide was more immunogenic than the 174-50 peptide by the intranasal (i.n.) route. Both chimeric peptides inhibited the binding of cholera toxin B subunit to GM1 ganglioside with the 50-174 peptide being more effective inhibitor than the 174-50 peptide. In addition, an effective priming of the immune system was achieved after the i.n. administration of immunogens. The observed unresponsiveness after the i.n. co-immunization with the 50-174 peptide and GM1 ganglioside emphasize the role of GM1 binding for the induction of an immune response after i.n. immunization.

Efficient binding to the MHC class I K(d) molecule of synthetic peptides in which the anchoring position 2 does not fit the consensus motif.

Peptides eluted from the MHC class I K(d) molecule are generally nonamers that display a strong preference for Tyr in position 2 and Ile or Leu in position 9. We investigated the binding ability of several synthetic peptides which did not fit this consensus motif. In our peptides, Tyr(2) was substituted by other amino acids, i.e. LeU, Ile or Met. These peptides were variants of the 252-260 K(d)-restricted peptide SYIPSAEKI derived from the Plasmodium berghei circumsporozoite protein. They bound to purified K(d) molecules in vitro with intermediate affinity. One of them was tested for in vivo stimulation of T cells and induced a cytotoxic response. These results demonstrate the importance of binding motif refinement to discover new binding characteristics and new ligands such as low-affinity peptides.

[Deep morphea-type lesions, first manifestations of lymphocytic lymphoma]. / Lésions à type de morphées profondes, premières manifestations d'un lymphome lymphocytique.

INTRODUCTION: The association between scleroderma and lymphoma is uncommon and can sometimes query an fortuitous association. More particular are observations where chronological and histological features show an evident link between a tumorous process and sclerosis. OBSERVATION: This case concerns a man 69 years old who was treated for one year for partial subcutaneous sclerosis present on his legs, thighs, fore-arms, and on the upper part of the trunk. No signs were present of visceral involvement evoking a systemic scleroderma and histology showed an intrication of a deep sclerosis, fasciitis and a tumoral lymphoma process. Diagnosis of lymphocytic lymphoma was confirmed. Initiating a chemotherapy (CHOP) allowed for a reduction of the sclerosis which didn't respond to the only corticotherapy. DISCUSSION: This observation can be linked to the association of fasciitis and lymphoma identified by Naschitz et al, but is different by: 1) the clinical aspect which correspond more to deep morphea lesions; 2) the histological link between sclerosis and lymphoma. This last point suggest that it is the tumoral population which have induced the sclerosis process linked to the secretion of pro-inflammatory factors. In adverse cases with longer delay between lymphoma and scleroderma this association may be fortuitous.

Purification of synthetic peptide libraries by affinity chromatography using the avidin-biotin system.

The specific interaction between biotin and avidin was exploited in the affinity purification of solid-phase synthesized peptide libraries. During peptide library synthesis, by means of the single-resin method in which coupling on variable positions is carried out using an equimolar mixture of amino acids, biotin was used to cap the unreacted amino groups remaining after coupling of the equimolar amino acid mixture. The following synthesis and deprotection procedures were performed as usual in tert,-butyloxycarbonyl chemistry. The purification of the peptide mixture containing N-biotinylated sequences was performed by affinity chromatography on an avidin-agarose column. The unwanted terminated sequences were retained in the avidin column while the purified peptide mixture was eluted as indicated by reverse-phase HPLC and MS analysis monitoring. The avidin column was regenerated and the biotinylated sequences were released under reversible denaturing conditions. The usefulness of biotinylation for peptide library purification is demonstrated here for the first time for a peptide mixture containing by-products that cannot be separated from the mixture by classical HPLC purification. This purification technique could be applied to all syntheses, presenting difficult reacting steps.

Influence of the conformation of a macromolecule on the generation of T-cell proliferative response. A study with model polypeptides.

To study the influence of the conformation of polypeptidic macromolecules on the generation of T-cell epitopes, sequential polypeptides with an octamer repeat unit were designed and synthesized. They adopt mainly unordered and alpha-helical conformations. Among these polypeptides, those containing proline are fully or partly unordered, and are more effective at inducing T-cell proliferation than a proline-free very stable alpha-helical polypeptide. This extremely stable alpha-helical conformation, probably stabilized by aggregation, would enhance its stability against proteolytic processing.

Oral immunization with a free peptide from cholera toxin: local protection and IgA production.

It is frequently of great benefit for good protection against pathogens to elicit a local immunization. For example the importance of antibacterial as well as antitoxin local secretory IgA, for protection against cholera, has been underlined in several studies. We have already reported that oral administration of the peptide corresponding to the 50-75 sequence of cholera toxin (CT) B subunit elicits serum antibodies neutralizing CT activity. In this study we demonstrate that IgA with specificity to CT are present in intestinal secretions of mice immunized orally with the P50-75 or P30-50 peptides of CT B subunit. In addition local protection is observed in the intestine of P50-75 orally immunized mice. These results point out the potential of synthetic peptides as immunogens at the mucosal level.

Synthesis of a new carrier for immunization: polytuftsin. Two examples of its use with peptides selected in the hepatitis B surface antigen.

Sequential poly(Arg-Thr-Lys-Pro) consisting mainly of the repeat of tuftsin Thr-Lys-Pro-Arg was synthesized by condensing the p-nitrophenyl ester of Arg(HCl)-Thr-Lys-(2-Cl-Z)-Pro in the presence of HOBt. Two haptenic sequences of the Pre-S region of hepatitis B virus antigen (10-26 and 39-55) were prepared by solid phase and coupled to polytuftsin via glutaraldehyde. The peptides, either free or coupled to polytuftsin, were administrated to mice and the antisera were assayed by ELISA. Coupling the peptides to the polypeptide significantly improved the anti-peptide antibody titer in Freund complete adjuvant or in NaCl 0.9%. Cross-reaction between antibodies induced by the peptides and the native protein was also improved. Polytuftsin alone is very poorly immunogenic.