[Paroxysmal dystonia and multiple sclerosis]. / Dystonie paroxystique et sclérose en plaques.

INTRODUCTION: Movement disorders are uncommon in multiple sclerosis, except for tremor. Patients rarely have paroxysmal dystonia (or tonic spasm), which can be the presenting manifestation of the disease. OBSERVATIONS: Two videotaped observations are presented. The first patient was a 27-year-old woman, treated for relapsing-remitting multiple sclerosis, who presented daily several short (<1minute) paroxysms of right hemibody dystonia. Brain MRI revealed several areas of cerebral demyelination, including the posterior limb of the left internal capsule with gadolinium enhancement. These events disappeared 7 days after corticosteroid infusion. The second patient was a 62-year-old man who presented brief episodes (<1minute) of daily painful left hemibody dystonia. Three months later, similar paroxysms affecting the right hemibody including the face occurred. At times, the two hemibodies were affected simultaneously. The brain MRI showed multiple areas of white matter hyperintensity, including two symmetrical areas in the posterior limb of the internal capsules. Multiple sclerosis was diagnosed on clinical, MRI and biological data. Four days after starting corticosteroids, these paroxysmal phenomena disappeared totally. CONCLUSION: Dystonia is an under-recognized aspect of paroxysmal events during multiple sclerosis. It might involve ephaptic transmission among abnormal demyelinated neurons; this ectopic excitation can arise at variable levels of the corticospinal tract, but the analysis of reported cases and those described in this study shows that impairment of the posterior limb of the internal capsule seems to be a prevalent topography. Inflammation is likely to play a role because steroids often improve these phenomena. In this article, we review the clinical aspects, pathophysiology and outcome of paroxysmal dystonia in multiple sclerosis.

[Pseudotumoral form of sclerochoroidal tuberculosis: a case report]. / Forme pseudotumorale de la tuberculose choroïdo-sclérale: à propos d'un cas.

The Authors report the US, CT and MR features of sclerochoroidal tuberculosis simulating a choroidal tumor in a 16 Year old female presenting with acute unilateral visual loss. Fundoscopic examination and fluorescein angiography showed a tumor at the posterior pole of the globe. CT and MRI showed the choroidal process and cerebral lesions suggestive of tuberculomas. The evolution was favorable with antituberculous treatment. Ocular tuberculosis is rare, especially the pseudotumoral form. It can simulate a choroidal tumor. Radiologists should be familiar with this appearance because the lesion is reversible with antituberculous treatment.

[Ocular relapse of acute lymphoblastic leukemia]. / Rechute oculaire de leucemie aigue lymphoblastique.

A bilateral leukemic hypopyon can be inaugural in the child's leukemia or reveal a relapse. A five years old child with acute lymphoblastic leukemia presented after 30 months of treatment a bilateral hypopyon. Anterior chamber paracentesis with cytological survey revealed leukemic cells and confirmed the ocular relapse. The treatment included the association of topical corticosteroids, chemotherapy and radiotherapy. This child died unfortunately 16 months later following a medullar relapse. We remind the different clinical aspects of leukemic invasion of the anterior segment and the therapeutic methods for this relapse.

Delineation of a neutralizing subregion within the immunodominant epitope (GH loop) of foot-and-mouth disease virus VP1 which does not contain the RGD motif.

The major immunogenic site of foot-and-mouth disease virus (FMDV) is contained in a disordered loop comprising residues 134-158 of capsid protein VP1, located on the surface of the viral particle. Peptides corresponding to this sequence generally elicit protective levels of neutralizing antibodies in guinea pigs. In some instances, however, the level of neutralizing antibodies is low although the level of antibodies against the peptide, determined by ELISA, is as high as that in the sera with high neutralizing antibody titres. In an attempt to ascertain the reason for this difference, we have synthesized on a cellulose membrane 10 overlapping decapeptides, offset by one residue, covering the segment 141-159 of VP1 of two viruses belonging to serotypes A12 and O1, and tested them with guinea pig antisera raised against peptide 141-159, VP1 and FMDV particles (SPOTscan method). With type A, some peptides which were strongly positive with highly neutralizing antisera did not include the RGD triplet located at residues 145-147. In contrast, antisera with low neutralization titres reacted only with decapeptides which included the RGD motif. Moreover, peptide 147-156 coupled to keyhole limpet haemocyanin, but not peptide 141-149 coupled to the same carrier, elicited high levels of neutralizing antibodies in guinea pigs. In the case of serotype O, highly neutralizing antisera to virus reacted in ELISA with peptides 141-150 (containing the RGD motif) and 135-144 (located upstream from the RGD motif). The results suggest that the RGD triplet is not an indispensable constituent of peptides able to elicit a neutralizing antibody response against the virus.

Solution structure of a retro-inverso peptide analogue mimicking the foot-and-mouth disease virus major antigenic site. Structural basis for its antigenic cross-reactivity with the parent peptide.

The antigenic activity of a 19-mer peptide corresponding to the major antigenic region of foot-and-mouth disease virus and its retro-enantiomeric analogue was found to be completely abolished when they were tested in a biosensor system in trifluoroethanol. This suggests that the folding pattern, which is alpha-helix in trifluoroethanol (confirmed by CD measurement), does not correspond to the biologically relevant conformation(s) recognized by antibodies. The NMR structures of both peptides were thus determined in aqueous solution. These studies showed that the two peptides exhibit similar folding features, particularly in their C termini. This may explain in part the cross-reactive properties of the two peptides in aqueous solution. However, the retro-inverso analogue appears to be more rigid than the parent peptide and contains five atypical beta-turns. This feature may explain why retro-inverso foot-and-mouth disease virus peptides are often better recognized than the parent peptide by anti-virion antibodies.

All-D peptides recognized by an anti-carbohydrate antibody identified from a positional scanning library.

Monoclonal antibodies recognize antigens with high affinity and specificity, but the structural basis for molecular mimicry remains unclear. It is often assumed that cross-reactive antigens share some structural similarity that is specifically recognized by a monoclonal antibody. Recent studies using combinatorial libraries, which are composed of millions of sequences, have examined antibody cross-reactivity in a manner entirely different from traditional epitope mapping approaches. Here, peptide libraries were screened against an anti-carbohydrate monoclonal antibody for the identification of peptide mimics. Positional scanning libraries composed of all-l or all-d hexapeptides were screened for inhibition of monoclonal antibody HGAC 39.G3 binding to an antigen displaying N-acetyl-d-glucosamine (GlcNAc) residues on a polyrhamnose backbone. Inhibitory activity by mixtures from the all-d hexapeptide library was greater than the activity from the all-l libraries. The most active d-amino acid residues defined in each of the six positions of the library were selected to prepare 27 different individual hexapeptides. The sequence Ac-yryygl-NH2 was specifically recognized by mAb HGAC 39.G3 with a relative affinity of 300 nM when measured in a competitive binding assay. The contributions to overall specificity of the residues of the all-d peptide (Ac-yryygl-NH2) in binding to mAb HGAC 39.G3 were examined with a series of truncation, l and d-amino acid substitution, and retro analogs. Dimeric forms of the all-d peptide were recognized with tenfold to 100-fold greater affinities relative to the monomer. The all-d peptide was found to inhibit mAb HGAC 39.G3 binding to an anti-idiotype antibody with approximately 1000-fold greater affinity than GlcNAc. As demonstrated here, the study of immune recognition using combinatorial chemistry may offer new insights into the molecular basis of cross-reactivity.

Identification of V3 loop-binding proteins as potential receptors implicated in the binding of HIV particles to CD4(+) cells.

The binding of human immunodeficiency virus (HIV) type 1 particles to CD4(+) cells could be blocked either by antibodies against the V3 loop domain of the viral external envelope glycoprotein gp120, or by the V3 loop mimicking pseudopeptide 5[Kpsi(CH2N)PR]-TASP, which forms a stable complex with a cell-surface-expressed 95-kDa protein. Here, by using an affinity matrix containing 5[Kpsi(CH2N)PR]-TASP and cytoplasmic extracts from human CEM cells, we purified three V3 loop-binding proteins of 95, 40, and 30 kDa, which after microsequencing were revealed to be as nucleolin, putative HLA class II-associated protein (PHAP) II, and PHAP I, respectively. The 95-kDa cell-surface protein was also isolated and found to be nucleolin. We show that recombinant preparations of gp120 bind the purified preparations containing the V3 loop-binding proteins with a high affinity, comparable to the binding of gp120 to soluble CD4. Such binding is inhibited either by 5[Kpsi(CH2N)PR]-TASP or antibodies against the V3 loop. Moreover, these purified preparations inhibit HIV entry into CD4(+) cells as efficiently as soluble CD4. Taken together, our results suggest that nucleolin, PHAP II, and PHAP I appear to be functional as potential receptors in the HIV binding process by virtue of their capacity to interact with the V3 loop of gp120.

The potential of retro-inverso peptides as synthetic vaccines.

Retro-inverso (RI) peptides, also called all-D-retro peptides, have been shown to mimic the antigenic and immunogenic properties of L-peptides successfully. RI peptides corresponding to the loop 141-159 of the VP1 protein of foot-and-mouth disease virus (FMDV) have been synthesized and used to immunize rabbits and guinea pigs. These peptides induced longer-lasting and higher antibody titres in immunized animals than did the corresponding L-peptides and the antibodies cross-reacted strongly with virus particles and with L-peptides. Antisera raised to RI peptides had in vitro virus neutralization titres equal to or better than those obtained after immunization with classical FMDV antigens and L-peptides. In view of their increased stability, RI peptides may overcome some of the shortcomings of synthetic viral vaccines based on L-peptides.

The potential of retro-inverso peptides as synthetic vaccines.

Retro-inverso peptides, also known as all-D-retro or retro-enantio peptides, are composed of D-amino acids assembled in the reverse order from that of the parent L-sequence. Since the orientation of the side-chains in a retro-inverso analogue is very similar to that in the parent L-peptide, this leads to a high level of antigenic cross-reactivity between the two peptides. The potential of retro-inverso peptides as synthetic vaccines has been investigated in the case of foot-and-mouth disease. A single inoculation of retro-inverso peptide corresponding to residues 141-159 of the VP1 protein of foot-and-mouth disease virus induced longer-lasting and higher antibody titres in immunised animals than the corresponding L-peptides. The antibodies cross-reacted strongly with virus particles and with L-peptides and conferred substantial protection in guinea-pigs challenged with the cognate virus. Retro-inverso peptides have considerable potential as synthetic vaccines, since their increased resistance to proteases may overcome one of the major drawbacks of classical L-peptide vaccines.

A retro-inverso peptide corresponding to the GH loop of foot-and-mouth disease virus elicits high levels of long-lasting protective neutralizing antibodies.

Peptides corresponding to the immunodominant loop located at residues 135-158 on capsid protein VP1 of foot-and-mouth disease virus (FMDV) generally elicit high levels of anti-peptide and virus-neutralizing antibodies. In some instances, however, the level of neutralizing antibodies is low or even negligible, even though the level of anti-peptide antibodies is high. We have shown previously that the antigenic activity of peptide 141-159 of VP1 of a variant of serotype A can be mimicked by a retro-inverso (all-D retro or retroenantio) peptide analogue. This retro-inverso analogue induced greater and longer-lasting antibody titers than did the corresponding L-peptide. We now show that a single inoculation of the retro-inverso analogue elicits high levels of neutralizing antibodies that persist longer than those induced against the corresponding L-peptide and confer substantial protection in guinea pigs challenged with the cognate virus. In view of the high stability to proteases of retro-inverso peptide analogues and their enhanced immunogenicity, these results have practical relevance in designing potential peptide vaccines.

Exploration of requirements for peptidomimetic immune recognition. Antigenic and immunogenic properties of reduced peptide bond pseudopeptide analogues of a histone hexapeptide.

We present a detailed analysis of the antigenic and immunogenic properties of a series of very stable peptidomimetics of a model hexapeptide corresponding to the C-terminal residues 130-135 of histone H3. Five pseudopeptide analogues of the natural sequence IRGERA were synthesized by systematically replacing, in each analogue, one peptide bond at a time by a reduced peptide bond Psi(CH2-NH). Three important features of the resulting analogues were examined. First, the analogues were tested in a biosensor system for their ability to bind monoclonal antibodies generated against the parent natural peptide, and their kinetic rate constants were measured. The results show that reduced peptide bond analogues can very efficiently mimic the parent peptide. The position of reduced bonds which were deleterious for the binding was found to depend on the antibody tested, and one monoclonal antibody recognized all five analogues. The equilibrium affinity constant toward reduced peptide bond analogues of four antibodies of IgG1 isotype induced against the parent hexapeptide was higher (up to 670 times) with certain analogues than toward the homologous peptide. Second, immunogenic properties of the five analogues were studied, and it was found that polyclonal antibodies induced against analogues in which Psi(CH2-NH) bonds were introduced between residues 130-131, 131-132, and 132-133 (R1-R2, R2-R3, and R3-R4) cross-reacted strongly with the cognate protein H3. Third, we tested the protease resistance of analogues. Altogether, the results provide a strong support for the potent applicability of reduced peptide bond pseudopeptides as components of synthetic vaccines and open a new field for the development of immunomodulatory agents.

Highly specific, cross-reactive sequences recognized by an anti-HBsAg antibody identified from a positional scanning synthetic combinatorial library.

The binding specificity of a monoclonal antibody (MAb 12) known to recognize the surface antigen of hepatitis B virus (HBsAg) was studied using a positional scanning synthetic combinatorial library. A hexapeptide library totaling more than 30 x 10(6) sequences, made up of 120 mixtures having a single position defined with individual amino acids and the remaining five positions composed of mixtures of amino acids, was screened by competitive enzyme-linked immunosorbent assay (ELISA). This led to the identification of Ac-STTSMM-NH2 (IC50 = 170 nM), which specifically inhibited the interaction between HBsAg and MAb 12. One of the most active individual mixtures from the library was Ac-XXXPXX-NH2; however, none of the individual peptides synthesized containing proline at the fourth position showed significant activity (IC50 > 100,000 nM). To identify the individual peptide(s) responsible for the activity of Ac-XXXPXX-NH2, a bidirectional iterative synthesis and selection process was carried out. A completely different but active peptide sequence was identified (Ac-SVGPPH-NH2, IC50 = 165 nM). The two different hexapeptide sequences were prepared as linear homo- and heterodimer peptides in an attempt to improve the antigenicity. Of the four different sequences prepared, one heterodimer (Ac-STTSMMGGGSVGPPH-NH2) was found by ELISA to have a 10-fold improvement in activity over the two individual hexapeptides, and was equal to the inhibitory activity of the protein antigen. The equilibrium affinity constant of the MAb 12 toward this heterodimer sequence was 50-fold higher than the protein antigen when measured in a biosensor system. Since motifs from these two hexapeptides, namely, -STTS- and -GP-, were located in the primary sequence of the protein (residues 114-120, subtype ad), overlapping hexapeptides of this region were synthesized and assayed. The most active hexapeptide, namely, Ac-TTSTGP-NH2 (IC50 = 2.3 microM), was 10-fold less active than either hexapeptide found from the library. Extending the specific motif sequences to eleven residues resulted in an analog (Ac-STTSTGPSRTC-NH2) having an equilibrium affinity constant similar to the heterodimer. The combined use of positional scanning libraries and the iterative synthesis and selection process in this study illustrates the power of these methods for the identification of novel peptides that inhibit anti-protein antibodies. These methods can be directly applied to the development of improved immunodiagnostics.

Inhibition of HIV infection by pseudopeptides blocking viral envelope glycoprotein-mediated membrane fusion and cell death.

The RP dipeptide motif is highly conserved in the third hypervariable region (V3 loop) of the extracellular envelope glycoprotein of different types of HIV isolates. In view of this, we have designed and synthesized a construction referred to as "template assembled synthetic peptide" (TASP), in which a lysine-rich short polypeptide was used as a template to covalently anchor arrays of tripeptides, such as RPR, RPK, or KPR. The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5(KPR)-TASP, molecules manifested maximum inhibitory activity on HIV infection with a 50% inhibitory concentration value of 1-5 microM, respectively. Structure and inhibitory-activity relationship studies using analogs of 5(KPR)-TASP indicated that the positively charged side chains of the K and R residues in the tripeptide molecules are critical for the optimal inhibitory activity of the pentavalent construct. Interestingly, replacement of L-amino acid residues by D-amino acids or reduction of the peptide bond between the first two amino acids of the tripeptide generated peptide-TASP analogs active at sub-microM, concentrations. The anti-HIV action of the peptide-TASP constructs is specific, since they inhibit infection of several types of CD4-expressing cells by HIV-1 Lai and HIV-2 EHO but not by the simian SIV-mac isolate. Our results suggest that these inhibitors block three post-CD4 binding functions of the HIV envelope glycoproteins, mediation of viral entry, syncytium formation, and triggering cell death by apoptosis. As the peptide-TASP derivatives with unnatural amino acid sequences in the tripeptide moiety retain full inhibitory activity, they should provide potent protease-resistant peptide inhibitors as potential therapeutic agents for treatment of AIDS patients.

Mimicry of viral epitopes with retro-inverso peptides of increased stability.

Two major limitations to the use of peptides as synthetic vaccines are their poor immunogenicity and low antigenic cross-reactivity with the epitopes of virus particles. Recently it has been shown that retro-inverso peptides corresponding to an immunodominant epitope of foot-and-mouth disease virus (FMDV) are able to mimic the structure and antigenic activity of natural L-peptides [1]. A series of L- and retro-inverso peptides of the loop 141-159 of the VP1 protein of FMDV has been synthesized. Antibodies to these peptides were produced by injecting rabbits with peptides covalently coupled to small unilamellar liposomes containing monophosphoryl lipid A as adjuvant. The retro-inverso peptides led to higher serum antibody titres which appeared earlier after the start of immunization and lasted longer than those found with L-peptides. Antibodies to retro-inverso peptides cross-reacted strongly with L-peptides and with virus particles, while guinea pig antisera to VP1 protein and virions cross-reacted strongly with the retro-inverso peptides. In view of their increased stability compared to natural L-peptides, retro-inverso peptidomimetics have considerable potential as synthetic viral vaccines.

Enhanced immunogenicity and cross-reactivity of retro-inverso peptidomimetics of the major antigenic site of foot-and-mouth disease virus.

Retro-inverso analogues of peptides corresponding to the major antigenic site 141-159 of VP1 from two foot-and-mouth disease virus variants have been synthesized and tested for their antigenic and immunogenic properties. Antibodies to the L- and retro-inverso peptides were produced by injecting rabbits with peptides covalently coupled to small unilamellar liposomes containing monophosphoryl lipid A as adjuvant. When compared to the antibody response raised against the L-peptides, the duration of the IgG response that was induced by the retro-inverso peptides was significantly longer and the titer of anti-peptide antisera was much higher. Antibodies to retro-inverso peptides cross-reacted equally well with the respective parent L-peptides. These results, obtained with a viral sequence which was found previously to represent a good candidate for possible vaccination, show that retro-inverso peptidomimetics could be useful for enhancing the immunogenicity of peptides.

Cross-reactivity of antibodies to retro-inverso peptidomimetics with the parent protein histone H3 and chromatin core particle. Specificity and kinetic rate-constant measurements.

A series of monoclonal antibodies has been generated against an hexapeptide of sequence IRGERA corresponding to the C-terminal residues 130-135 of histone H3 and three analogues of this model peptide. The analogues correspond to the D-enatiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural amide peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retro-inverso-peptide. Monoclonal antibodies were generated from mice immunized with the analogues coupled to neutral small unilamellar liposomes containing monophosphoryl lipid A as adjuvant. The reactivity of antibodies with the four analogues and with the parent protein H3 was studied in enzyme-linked immunosorbent assay and in a biosensor system. The equilibrium affinity constant (Ka) toward the retro-inverso-peptide of two out of three antibodies of IgG1 isotype induced against the L-hexapeptide was 7-75-fold higher than toward the homologous L-peptide. The range of Ka values of four antibodies of IgG1 and IgG2a isotypes generated against the retro-inverso-peptide was 0.6-1.9 x 10(9) M-1 for both the retro-inverso- and L-peptides. Furthermore, antibodies to the L- and retro-inverso-peptides cross-reacted strongly (in some cases better than with the homologous peptide) with the parent histone H3 and with chromatin subunits containing H3. The results are thus promising in respect to the potential use of retro-inverso-analogues, which are particularly stable, in the design of much more potent synthetic vaccines or to generate antibody probes.

Synthesis and antigenic properties of reduced peptide bond pseudopeptide analogues of a histone H3 hexapeptide.

Pseudopeptide analogues of the C-terminal hexapeptide of histone H3 (-Ile-Arg-Gly-Glu-Arg-Ala-OH) were obtained by systematically replacing, in each analogue, one peptide bond at a time by a reduced peptide bond psi (CH2-NH). The resulting analogues were then examined, in ELISA and in the BIAcore system, for their ability to bind polyclonal and monoclonal antibodies generated against the parent natural peptide and the protein. The comparative results show that reduced bond pseudopeptide analogues can mimic the parent peptide. These results present the first unequivocal example for the potent applicability of reduced peptide bond pseudopeptides in the immunological field.

Antigenic mimicry of natural L-peptides with retro-inverso-peptidomimetics.

Three analogues of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 were synthesized, and their antigenicity, immunogenicity, and resistance to trypsin were compared to those of the natural L-peptide. The three analogues correspond to the D-enantiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retro-inverso-peptide. Antibodies to the four peptide analogues were produced by injecting BALB/c mice with peptides covalently coupled to small unilamellar liposomes containing monophosphoryl lipid A. Each of the four peptide analogues induced IgG antibodies of various subclasses. The IgG3 antibodies reacted similarly with the four analogues, whereas antibodies of the IgG1, IgG2a, and IgG2b isotypes showed strong conformational preferences for certain peptides. The retro-inverso-peptide IRGERA mimicked the structure and antigenic activity of the natural L-peptide but not of the D- and retro-peptides, whereas the retro-peptide IRGERA mimicked the D-peptide but not the L- and retro-inverso-peptides. The equilibrium affinity constants (Ka) of three monoclonal antibodies generated against the L- and D-peptides with respect to the four peptide analogues were measured in a biosensor system. Large differences in Ka values were observed when each monoclonal antibody was tested with respect to the four peptides. The use of retro-inverso-peptides to replace natural L-peptides is likely to find many applications in immunodiagnosis and as potential synthetic vaccines.

Antigenicity and immunogenicity of modified synthetic peptides containing D-amino acid residues. Antibodies to a D-enantiomer do recognize the parent L-hexapeptide and reciprocally.

The effect of introducing D-amino acid residues in an hexapeptide was examined both at the antigenic and immunogenic levels. A series of D-analogues of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 were produced. Four analogues contained a single change of an L-residue by the corresponding enantiomer, one peptide contained two D-residues and another one contained only D-residues (D-enantiomer). A peptide analogue was also synthesized in which the 2 Arg residues were replaced by Lys residues. The parent peptide and peptide analogues were injected into mice after covalent coupling to small unilamellar liposomes containing monophosphoryl lipid A as adjuvant. The substitution of L-Arg131 to Lys or D-Arg was found to change neither the antigenic nor immunogenic properties of the resulting peptides. In contrast, the substitution of Glu133, Arg134, and Ala135 by the respective enantiomers drastically altered the antigenicity of the modified peptides. Each of the six D-analogues induced an immune response with an unusually high level of IgG3 antibodies. The D-enantiomer produced IgG3 antibodies which reacted with the homologous peptide as well as with the all L-peptide and the parent protein H3 in solution but not with analogues containing one or two D-residues only. IgG3 antibodies produced against the all L-peptide reacted with the free all D-peptide but not with the other analogues containing D-residues in position 133, 134, and 135.