Adhesion of the ulcerative pathogen Mycobacterium ulcerans to DACC-coated dressings.

OBJECTIVE: Mycobacterium ulcerans is the causative agent of Buruli ulcer disease, the third most common mycobacteriosis after tuberculosis and leprosy and an emerging public health threat in sub-Saharan Africa. The bacteria produce a diffusible cytotoxin called mycolactone, which triggers the formation of necrotic lesions in cutaneous and subcutaneous tissues. The principal aim of this study was to characterise the cell surface hydrophobicity of Mycobacterium ulcerans and determine if bacteria bind to dialkyl carbamoyl chloride (DACC)-coated dressings through hydrophobic interactions in vitro. Since mycolactone displays hydrophobic groups, a secondary aim was to compare mycolactone binding to hydrophobic and standard dressings. METHODS: We used hydrophobic interaction chromatography to evaluate the cell surface hydrophobicity of Mycobacterium ulcerans, compared to that of other microorganisms colonising wounds. The binding of Mycobacterium ulcerans bacteria to DACC-coated and control dressings was then assessed quantitatively by measurement of microbial adenosine triphosphate (ATP), while that of mycolactone was evaluated by fluorescence spectroscopy. RESULTS: Compared to Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, Mycobacterium ulcerans displayed the highest cell surface hydrophobicity, irrespective of the bacterial production of mycolactone. Mycobacterium ulcerans bacteria bound to DACC-coated dressings [corrected] better than untreated controls. Mycolactone did not bind stably to hydrophobic, nor standard dressings, in the conditions tested. CONCLUSION: Retention of Mycobacterium ulcerans and other wound pathogens to DACC-coated dressings may help reduce the bacterial load in Buruli ulcers and thereby improve healing. Dressings efficiently capturing mycolactone may bring an additional clinical benefit, by accelerating the elimination of the toxin during the course of antibiotic treatment.

Microstructural characterization of Ti-6Al-4V alloy subjected to the duplex SMAT/plasma nitriding.

In this study, microstructural characterization of Ti-6Al-4V alloy, subjected to the duplex surface mechanical attrition treatment (SMAT)/nitriding treatment, leading to improve its mechanical properties, was carried out through novel and original samples preparation methods. Instead of acid etching which is limited for morphological characterization by scanning electron microscopy (SEM), an original ion polishing method was developed. Moreover, for structural characterization by transmission electron microscopy (TEM), an ion milling method based with the use of two ions guns was also carried out for cross-section preparation. To demonstrate the efficiency of the two developed methods, morphological investigations were done by traditional SEM and field emission gun SEM. This was followed by structural investigations through selected area electron diffraction (SAED) coupled with TEM and X-ray diffraction techniques. The results demonstrated that ionic polishing allowed to reveal a variation of the microstructure according to the surface treatment that could not be observed by acid etching preparation. TEM associated to SAED and X-ray diffraction provided information regarding the nanostructure compositional changes induced by the duplex SMAT/nitriding process.

Surface nanocrystallization of Ti-6Al-4V alloy: microstructural and mechanical characterization.

In this study, microstructural and mechanical properties of Ti-6Al-4V alloy, before and after the SMA treatment (SMAT) as well as the duplex SMAT/Nitriding process at different treatment conditions, were investigated in order to deepen the knowledge of these properties for biomedical devices. For that purpose, tribological (wear resistance, coefficient of friction) and mechanical (Vickers microhardness) tests were performed. To carry out the microstructural and surface topographical characterization of the samples, the scanning electron microscopy (SEM) and the 3D-SEM reconstruction from stereoscopic images have been used. By means of profiles deduced from the 3D images, the surface roughness has been calculated. The obtained results allowed to find an interesting SMAT condition which, followed by nitriding at low temperature, can greatly improve tribological and mechanical properties of Ti-6Al-4V alloy. It was also shown from SEM characterization and the original method of 3D-SEM reconstruction, that SMAT can reduce the machined grooves and consequently the roughness of the samples decreases. Moreover, we demonstrated, for the first time, that instead of usual etching method, the ionic polishing allowed to reveal the grains, the grain boundaries and the twins as well as the surface nanocrystalline layer generated by SMAT. Thus, the thickness of the SMATed layer decreases with the nitriding temperature, whereas the surface grain size increases.

Lipids of pathogenic Mycobacteria: contributions to virulence and host immune suppression.

Mycobacteria are characterized by a complex cell wall, the lipid nature of which confers to the bacilli resistance to drying, acid or alkaline conditions, and to chemical disinfectants and therapeutic agents. Pathogenic species, such as Mycobacterium tuberculosis, M. leprae and M. ulcerans, have evolved various strategies to establish residence in their hosts and provoke long-term infections. There is mounting evidence that the unique lipids composing their envelopes, strategically located at the host-pathogen interface, contribute to their escape from immune surveillance. Here, the chemical structure, host cell receptors and biological actions of this emerging class of mycobacterial virulence factors are reviewed.

Priming by DNA immunization augments protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin against tuberculosis.

Sequential immunization with mycobacterial antigen Ag85B-expressing DNA and Mycobacterium bovis bacille Calmette-Guerin (BCG) was more effective than BCG immunization in protecting against Mycobacterium tuberculosis infection. Depletion of the CD8(+) T cells in the immunized mice impaired protection in their spleens, indicating that this improved efficacy was partially mediated by CD8(+) T cells.

Dendritic cells infected with Mycobacterium bovis bacillus Calmette Guerin activate CD8(+) T cells with specificity for a novel mycobacterial epitope.

Although CD4(+) T cells are essential for protective immunity against Mycobacterium tuberculosis infection, recent reports indicate that CD8(+) T cells may also play a critical role in the control of this infection. However, the epitope specificity and the mechanisms of activation of mycobacteria-reactive CD8(+) T cells are poorly characterized. In order to study the CD8(+) T cell responses to the model mycobacterial antigen, MPT64, we used recombinant vaccinia virus expressing MPT64 (VVWR-64) and a panel of MPT64-derived peptides to establish that the peptide MPT64(190-198) contains an H-2D(b)-restricted CD8(+) T cell epitope. A cytotoxic T lymphocyte response to this peptide could be demonstrated in M. bovis bacillus Calmette Guerin (BCG)-infected mice following repeated in vitro stimulation. When bone marrow-derived dendritic cells (DC) were infected with BCG, the expression of MHC class I molecules by DC was up-regulated in parallel with MHC class II and B7-2, whereas CD1d expression level was not modified. Moreover, BCG-infected DC activated MPT64(190-198)-specific CD8(+) T cells to secrete IFN-gamma, although with a lower efficacy than VVWR-64-infected DC. The production of IFN-gamma by MPT64(190-198)-specific CD8(+) T cells was inhibited by antibodies to MHC class I, but not to CD1d. These data suggest that mycobacteria-specific CD8(+) T cells are primed during infection. Therefore, anti-mycobacterial vaccine strategies targeting the activation of specific CD8(+) T cells by DC may have improved protective efficacy.

Stimulation of dendritic cells via CD40 enhances immune responses to Mycobacterium tuberculosis infection.

The resolution of pulmonary tuberculosis (TB) critically depends on the development of the Th1 type of immune responses, as exemplified by the exacerbation of TB in IL-12-deficient mice. Therefore, vaccination strategies optimizing IL-12 production by antigen-presenting cells (APC) in response to mycobacteria may have enhanced protective efficacy. Since dendritic cells (DC) are the critical APC for activation of CD4+ and CD8+ T cells, we examined whether stimulation of Mycobacterium bovis bacillus Calmette Guérin (BCG)-infected DC via CD40 increased their ability to generate Th1-oriented cellular immune responses. Incubation of DC with an agonistic anti-CD40 antibody activated CD40 signaling in DC, as shown by increased expression of major histocompatibility complex class II and costimulatory molecules, mRNA production for proinflammatory cytokines and interleukin 12 (IL-12) p40. This activation pattern was maintained when DC were stimulated with anti-CD40 antibody and infected with BCG. Importantly, CD40-stimulated BCG-infected DC displayed increased capacity to release bioactive IL-12 and to activate gamma interferon (IFN-gamma) producing T cells in vitro. Moreover, when C57BL/6 mice were immunized with these DC and challenged with aerosol Mycobacterium tuberculosis, increased levels of mRNA for IL-12 p40, IL-18, and IFN-gamma were present in the draining mediastinal lymph nodes. However, the mycobacterial burden in the lungs was not reduced compared to that in mice immunized with BCG-infected non-CD40-stimulated DC. Therefore, although the manipulation of DC via CD40 is effective for enhancing immune responses to mycobacteria in vivo, additional strategies are required to increase protection against virulent M. tuberculosis infection.

Interaction of dendritic cells with mycobacteria: where the action starts.

Dendritic cells (DC) are the major antigen-presenting cells in the induction of cellular responses to intracellular pathogens, such as mycobacteria. Recent studies have shown that they also play a critical role in the regulation of immune responses. The interaction of DC with microbial antigens may be the controlling factor in the development of a Th1-orientated protective immunity. Analysis of the innate response of DC to mycobacteria and the involvement of the DC receptors in antigen recognition have highlighted the pivotal role of these cells in T-cell activation. Mycobacteria-infected DC have an enhanced capacity to release pro-inflammatory cytokines and chemokines and are potent inducers of interferon-gamma-producing cells in vivo. Therefore, DC manipulation for maximal antigen presentation and Th1 cytokine production may form the basis of a new generation of vaccines, with improved efficacy against mycobacterial infections.

Combining phage display and molecular modeling to map the epitope of a neutralizing antitoxin antibody.

Crotoxin is a potent presynaptic neurotoxin from the venom of the rattlesnake Crotalus durissus terrificus. It is composed of the noncovalent and synergistic association of a weakly toxic phospholipase A2, CB, and a nontoxic three-chain subunit, CA, which increases the lethal potency of CB. The A-56.36 mAb is able to dissociate the crotoxin complex by binding to the CA subunit, thereby neutralizing its toxicity. Because A-56.36 and CB show sequence homology and both compete for binding to CA, we postulated that A-56.36 and CB had overlapping binding sites on CA. By screening random phage-displayed libraries with the mAb, phagotopes bearing the (D/S)GY(A/G) or AAXI consensus motifs were selected. They all bound A-56.36 in ELISA and competed with CA for mAb binding, although with different reactivities. When mice were immunized with the selected clones, polyclonal sera reacting with CA were induced. Interestingly, the raised antibodies retained the crotoxin-dissociating effect of A-56.36, suggesting that the selected peptides may be used to produce neutralizing antibodies. By combining these data with the molecular modeling of CA, it appeared that the functional epitope of A-56.36 on CA was conformational, one subregion being discontinuous and corresponding to the first family of peptides, the other subregion being continuous and composed of amino acids of the second family. Phage-displayed peptides corresponding to fragments of the two identified regions on CA reacted with A-56.36 and with CB. Our data support the hypothesis that A-56.36 and CB interact with common regions of CA, and highlight residues which are likely to be critical for CA-CB complex formation.

Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis.

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.

Molecular mimicry between a monoclonal antibody and one subunit of crotoxin, a heterodimeric phospholipase A2 neurotoxin.

Crotoxin is a heterodimeric phospholipase A2 neurotoxin formed by the non-covalent association of an acidic and non-toxic subunit, CA, and a basic and weakly toxic phospholipase A2, CB. The two subunits behave in a synergistic manner. CA enhances the lethal potency of CB by increasing its selectivity of action. The mAb A-56.36, directed against the non-toxic subunit CA, was previously shown to neutralize crotoxin toxicity by dissociating the crotoxin complex. In the present report, a polypeptide sequence similarity was observed between some CDRs of mAb A-56.36 and two regions of CB (pos. 60-80 and 95-110). Phage displayed peptides corresponding to VH2 and VH3 of mAb A-56.36 and to their homologous sequences in CB bind CA to different extents. This observation shows that mAb A-56.36 interacts with a region of CA involved in its interaction with CB, therefore mimicking the binding of CB to CA. A similar approach was used to determine the regions of ammodytoxin A and of agkistrodotoxin, two phospholipase A2 neurotoxins similar to CB, which are involved in the formation of heterocomplexes with CA. The analysis of these data contributes to the determination of stretches of amino acids which could constitute the paratope of mAb A-56.36, as well as the region of association of CB with CA in crotoxin.

Protection against aerosol Mycobacterium tuberculosis infection using Mycobacterium bovis Bacillus Calmette Guérin-infected dendritic cells.

In the lung, dendritic cells (DC) are key antigen-presenting cells capable of triggering specific cellular responses to inhaled pathogens, and thus, they may be important in the initiation of an early response to mycobacterial infections. The ability of DC to enhance antigen presentation to naive T cells within the lungs was characterized with respect to Mycobacterium bovis Bacillus Calmette Guérin (BCG) vaccination against M. tuberculosis infection. In vitro derived DC were infected with BCG, which induced their maturation, as shown by the increased expression of MHC class II antigens, CD80 and CD86 co-stimulatory molecules. The synthesis of mRNA for IL-1, IL-6, IL-12, IL-10 and IL-1 receptor antagonist was also enhanced. When administered intratracheally in mice, infected DC induced a potent T cell response and the production of IFN-gamma to mycobacterial antigens in the mediastinal lymph nodes, leading to a significant protection against aerosol M. tuberculosis infection. Intriguingly, although the vaccination schedule for BCG-infected DC was much shorter than subcutaneous BCG vaccination (7 days as compared to 100 days), both types of vaccination showed similar levels of protection. These data confirm that DC can be potent inducers of a cellular immune response against mycobacteria and support the concept of combining DC strategies with mycobacterial vaccines for protective immunity against tuberculosis.

Phage-displayed mimotopes elicit monoclonal antibodies specific for a malaria vaccine candidate.

The phage-displayed peptide CGRVCLRC (C15) has been isolated from a random library by affinity screening with the D14-3 monoclonal antibody, which was raised to the 42 kDa C-terminal fragment of the major merozoite surface protein 1 of Plasmodium vivax (Pv42). In order to investigate the use of such mimotopes as possible vaccine components, we studied the antibody response in Biozzi mice immunized with C15. High titers of antibodies cross-reacting with Pv42 were generated and the IC50 of all immune sera were in the 5 x 10(-9) M range. Two monoclonal antibodies that specifically bind the Pv42 fragment were isolated. Although these mAbs had a lower affinity for Pv42 when compared to D14-3, they reproduced the cross-reactivity of D14-3 with the equivalent protein in P. cynomolgi, a close relative of P. vivax. DNA sequence analysis showed similarities between the germline genes and the canonical CDR conformations of all three antibodies, but molecular modeling failed to reveal common structural features of their paratopes that could account for their cross-reacting patterns. These data demonstrate that mimotopes selected from random repertoires do not necessarily represent structural equivalents of the original antigen but provide functional images that could replace it for vaccine development.

Phage-displayed and soluble mouse scFv fragments neutralize rabies virus.

A phage-display technology was used to produce a single-chain Fv antibody fragment (scFv) from the 30AA5 hybridoma secreting anti-glycoprotein monoclonal antibody (MAb) that neutralizes rabies virus. ScFv was constructed and then cloned for expression as a protein fusion with the g3p minor coat protein of filamentous phage. The display of antibody fragment on the phage surface allows its selection by affinity using an enzyme-linked immunosorbent assay (ELISA); the selected scFv fragment was produced in a soluble form secreted by E. coli. The DNA fragment was sequenced to define the germline gene family and the amino-acid subgroups of the heavy (VH) and light (VL) chain variable regions. The specificity characteristics and neutralization capacity of phage-displayed and soluble scFv fragments were found to be identical to those of the parental 30AA5 MAb directed against antigenic site II of rabies glycoprotein. Phage-display technology allows the production of new antibody molecule forms able to neutralize the rabies virus specifically. The next step could be to engineer and produce multivalent and multispecific neutralizing antibody fragments. A cocktail of multispecific neutralizing antibodies could contain monovalent, bivalent or tetravalent scFv fragments, for passive immunoglobulin therapy.

Biologically active human anti-crotoxin scFv isolated from a semi-synthetic phage library.

BACKGROUND: The display of repertoires of antibody fragments on the surface of filamentous bacteriophages offers a new way of making antibodies with predefined binding specificities. OBJECTIVES: Here we explored the use of this technology to find human antibodies with biological properties. Phage-scFv specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus, were isolated from a 'single pot' repertoire of more than 10(8) clones made in vitro from human V gene segments [1]. The crotoxin molecule is composed of two noncovalently linked subunits: a basic and weakly toxic phospholipase A2 (PLA2) called component B (CB) and an acidic, nonenzymatic and nontoxic subunit called component A (CA). CA is able to increase the toxicity as well as the specificity of action of CB simultaneously reducing its enzymatic activity. STUDY DESIGN: Two clones were isolated (4-21 and 5-3-1) which are specific of the basic subunit CB, but of a moderate affinity (about 10(-7) M). Clones 4-21 and 5-3-1 have different amino acid sequences and different effects on CB properties suggesting that they are raised against different CB epitopes. Purely cholinergic synaptosomes isolated from Torpedo electric organs provide a suitable model to study the presynaptic effects of crotoxin. In this model, CB was shown to induce a larger acetylcholine release than crotoxin. RESULTS: A dose-dependent increase of acetylcholine release was observed when crotoxin was incubated with increasing amounts of phage-scFv 4-21. This clone was also shown to increase the enzymatic activity of crotoxin. These observations suggest that phage-scFv might dissociate the complex CA-CB. It could be therefore a neutralizing antibody since CB is much less toxic than crotoxin. This shows that 'single pot' libraries are capable of providing not only immunochemical reagents of high specificity but also biological reagents of high quality. The use of this library appears to open new possibilities for immune passive therapy.

Mutations in the MHC class I-like candidate gene for hemochromatosis in French patients.

A candidate gene for hemochromatosis has recently been localized on the short arm of chromosome 6, about 4 megabases telomeric to the major histocompatibility complex. It encodes a protein that exhibits significant similarity to the HLA class I molecules and can be provisionally designated HLA-hc. Genotype analysis of 94 hemochromatosis patients living in France and a similar number of controls confirms that the disease is strongly associated with homozygosity at nucleotide 845 (72% of the patients and none of the controls carry two copies of the 845A variant). The data are consistent with hemochromatosis being a heterogeneous disease: about 79% of the cases in this sample would be caused by a defect in HLA-hc and 21% by an unrelated mechanism. A second variant (187 G) enriched on patient chromosomes that do not carry the 845A mutation might influence the affinity of a ligand for HLA-hc; the exact nature of this ligand remains to be discovered. The 845A variant is the best genetic marker for the disease identified to date, and the detection of 845A homozygosity should now permit diagnosis of a readily curable disease and the prevention of sometimes deadly complications in at least 72% of the patients.

Reproducing the immune response against the Plasmodium vivax merozoite surface protein 1 with mimotopes selected from a phage-displayed peptide library.

We have used phage display technology to identify peptides binding D14-3, a monoclonal antibody raised against the M(r) 42,000 C-terminal fragment of Plasmodium vivax merozoite surface protein 1 (PvMSP1). By screening a constrained hexapeptide library, seven independent clones binding D14-3 were isolated. The reactivity of D14-3 for these peptides was lower than for the natural antigen and the antibody binding was strictly associated with the viral context and the peptide conformation. Sequence analysis showed that five of them shared homology with the M(r) 42,000 C-terminal fragment (Pv42) and therefore appears to identify the D14-3 epitope. However, the other two peptides, while related to each other, did not correspond to any sequence in the Pv42 molecules. To evaluate their immunological interest, these phagotopes were injected into mice belonging to Balb/c, IC57BI/6 and Biozzi strains. All animals developed a strong immune response against phage particles but only Biozzi mice produced antibodies cross-reacting with Pv42. All phagotopes in Biozzi mice elicited a specific response against Pv42, even those sharing no sequence similarity with the antigen. Moreover, the avidities of these immune sera and the polyclonal response against Pv42 were comparable, suggesting phagotopes could be used as components of a subunit vaccine based on the C-terminal fragment of MSP1.

An attempt to correlate cattle breed origins and diseases associated with or transmitted by the tick Amblyomma variegatum in the French West Indies.

By using biological data and historical research, we have tried to explain the difference between resistance and susceptibility to the diseases transmitted (cowdriosis) or associated (dermatophilosis) with the tick Amblyomma variegatum, in two cattle breeds of the French West Indies: the Creole crossbred cattle of Guadeloupe and the Brahman zebu cattle of Martinique. Have been studied the polymorphisms of 5 independent genetic systems (erythrocytic haemoglobin, serum albumin and transferrin, the class I region of the BoLA complex and the gamma S crystallin gene) in different breeds comprising Bos taurus cattle of Europe and Africa, Bos indicus of West and East Africa, as well as the Brahman of Martinique and the Creole crossbred of Guadeloupe. By comparing the different allele frequencies of these 5 non related polymorphic loci and by using the two different mathematical matrices of NEI and of CAVALLI-SFORZA, have been established the genetic distances between these breeds. It appears clearly that the Creole cattle of Guadeloupe are in an intermediate position between the Bos taurus N'Dama breed of West Africa and two Bos indicus zebu breeds, namely the West African Sudan zebu and the Brahman. Thanks to studies of different archieves in the Caribbean and in Europe, historical evidence have been accumulated on the geographical origins and on the chronology of the establishment of Creole and Brahman cattle in the French West Indies. The high resistance of the Creole cattle of Guadeloupe to diseases associated with or transmitted by the "Senegalese" tick Amblyomma variegatum seems to be due to the inheritance of a pool of genes from West African cattle and more particularly from the N'Dama breed.(ABSTRACT TRUNCATED AT 250 WORDS)

Lactate dehydrogenase (LDH) activity of the cultured eukaryotic cells as marker of the number of dead cells in the medium [corrected].

One significant problem in monitoring a culture's evolution is to assess change in cell viability. We have demonstrated that LDH release could be a good indicator of cellular damage of many cell lines, especially during shear stress or sonication. Moreover, we have found a significant correlation between the number of dead cells, determined by Trypan Blue staining, and LDH activity measurements in the supernatant of hybridoma strains, whatever the culture conditions. We have also shown that when viability is still near 100% no LDH is released even at high cell concentrations. Therefore, LDH should serve as a potential marker of cell injury and death.