Field evaluation of rapid diagnostic tests for meningococcal meningitis in Niger.

OBJECTIVE: To evaluate dipstick rapid diagnostic tests (RDTs) for meningococcal meningitis in basic health facilities. METHODS: Health facility staff received a one-day training. During the meningitis season, they performed RDTs on cerebrospinal fluid (CSF) specimens from suspected cases of meningitis. A frozen aliquot of CSF was later tested using polymerase chain reaction (PCR) to establish the reference diagnosis. RDTs used in health facilities were archived to allow checking the concordance between reported diagnosis and observed results. Reported diagnosis was also compared to PCR diagnosis. A second RDT was performed on each CSF specimen at the reference laboratory. RESULTS: Using RDTs, health facilities reported 382 negative results (73.9%), 114 NmA (22.1%), 12 NmW135 (2.3%) and nine uninterpretable results (1.7%), the latter corresponding to the misuse of a reagent by three agents. The agreement between reported diagnosis and archived dipsticks was excellent (kappa = 0.98). The agreement between PCR diagnosis and reported RDTs results was strong (kappa = 0.82). In health facilities, the sensitivity of RDTs for N. meningitidis A was Se = 0.91. The kappa coefficient measuring the agreement between RDTs operated in the reference laboratory and RDTs operated in health facilities was kappa = 0.78. CONCLUSION: We confirmed that dipstick RDTs to identify N. meningitidis serogroups A, C, W135 and Y can be reliably operated by non-specialized staff in basic health facilities. RDTs proved very useful to recommend vaccination in NmA epidemics, and also to avoid vaccination in epidemics due to serogroups not included in vaccines (NmX).

Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix.

Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule.Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated.

Immunohistochemical localization of dentin matrix protein 1 in human dentin.

Dentin matrix protein 1 (DMP1) is a non-collagenous matrix protein with a recognized role in the formation of mineralized tissues such as dentin. The aim of this study was to analyze the distribution of DMP1 in human dentin by means of immunofluorescence and high-resolution immunogold labeling. Fully developed, sound human dentin specimens were submitted to fluorescence labeling and post-embedding immunolabeling techniques with a rabbit polyclonal antihuman DMP1 antibody followed by corresponding fluorochrome-conjugated or gold-conjugated secondary antibodies. Both immunofluorescence and immunogold labeling showed an intense labeling associated with the peritubular dentin. In addition, at the ultrastructural level, there was also a moderate and diffuse immunoreaction over intertubular dentin, and a weak labeling within predentin which increased in density towards the mineralization front. This study suggests that in adult human teeth, like in rodents, DMP1 is prevalently concentrated at the level of peritubular dentin and this feature is preserved also in fully developed-teeth. These data are consistent with what has been observed in rodents and suggest that DMP1 plays a role in maintenance of the dentin tubular space.

Approach to the mechanism of Zajdela's hepatoma cell growth inhibition induced by concanavalin A and phytohaemagglutinin.

Cell growth of tumour ascites cells was inhibited by concanavalin A, phytohaemagglutinin and Ricinus lectin at 2-100 micrograms/ml. As expected, the Ricinus lectin inhibited the protein synthesis estimated by leucine incorporation and decreased thymidine incorporation, whereas concanavalin A and phytohaemagglutinin stimulate the uptake and the incorporation of both leucine and thymidine, and thus, synthesis of protein and DNA. These results suggest that different mechanisms are involved in the hepatoma cell growth inhibition by the lectins. This difference was not related to the kinetic characteristics of the lectin interactions with the cells which represent a first and necessary step. It was showed that concanavalin A and phytohaemagglutinin as well as chloroquine inhibited the 14C-labelled asialofetuin degradation. We can conclude that Ricinus lectin present a toxic effect whereas both concanavalin A and phytohaemagglutinin show an anti-protease activity.

Biosynthesis of high molecular weight polylactosamine-type glycopeptides in rat Zajdela hepatoma ascites cells.

The first steps of the biosynthetic pathway of high molecular weight polylactosamine-type glycopeptides from rat Zajdela hepatoma cells were studied by pulse-chase experiments, biochemical analysis and by inhibition of N-glycosylation. It is clear that this process involves firstly the transfer of a lipid-linked high-mannose oligosaccharide precursor to a protein moiety in a similar way to that of N-linked glycopeptides of a more common size range according to the classical 'cycle of dolichol'. In the presence of enzymes which are inhibitors of the processing of high-mannose oligosaccharide chains, this class of oligosaccharides was considerably increased, whereas polylactosamine chains and lower complex N-linked glycopeptides were concomitantly decreased in the same kinetics and the same ratio. As expected in the presence of N-methyldeoxynojirimycin, which is an alpha-glucosidase inhibitor, high-mannose oligosaccharides remained glycosylated and are mostly of the Glc1-3Man9GlcNAc type. In the presence of swainsonine, which is an alpha-mannosidase (EC 3.2.1.24) inhibitor, these chains were devoid of glucose residues. In addition, some chains displayed hybrid structures. It appears, therefore, that the first steps of the biosynthesis of polylactosamine-type and N-linked oligosaccharides of a more common size range proceed similarly and that differences between their biosynthetic pathways occur during the elongation phase, which leads to their final respective structures. Glycopeptides prepared from the cell surface by mild trypsin treatment as well as from entire cells, previously treated or not by processing inhibitors, display the same gel filtration patterns indicating that modifications in protein glycosylation do not prevent glycoprotein insertion into the cell membrane.

The 7alpha-hydroxysteroids produced in human tonsils enhance the immune response to tetanus toxoid and Bordetella pertussis antigens.

Human tonsils were assessed for their ability to 7alpha-hydroxylate pregnenolone (PREG), dehydroepiandrosterone (DHEA) and 3-epiandrosterone (EPIA). Both 7alpha-hydroxy-DHEA and 7alpha-hydroxy-EPIA were produced by homogenates of either whole tonsils or of lymphocyte-depleted tonsil fractions. In contrast, isolated lymphocytes were found to be unable to carry out 7alpha-hydroxylation. When co-cultures of tonsil-derived T and B lymphocytes were set up under stimulatory conditions, IgGs were released in the supernatants and could be quantitated, and immunomodulating properties of different steroids were monitored. When PREG was added to a mixture of tonsil-derived B and T lymphocytes, a decrease of non-specific and specific IgG was observed. An increase in specific anti-tetanus toxoid and anti-Bordetella pertussis antigen IgGs was obtained with either 1 microM 7alpha-hydroxy-DHEA or 1 microM 7alpha-hydroxy-EPIA. In contrast, DHEA and EPIA were unable to trigger such an effect. When cultures of isolated tonsillar B cells were used, none of the steroids tested showed significant effects on specific IgG productions. These data led to the conclusion that human tonsillar cells transform DHEA and EPIA, but not PREG, into 7alpha-hydroxylated metabolites. These metabolites could act on target tonsillar T lymphocytes which in turn act upon B lymphocytes for increasing specific IgG production.

Crystal structure of a Fab complex formed with PfMSP1-19, the C-terminal fragment of merozoite surface protein 1 from Plasmodium falciparum: a malaria vaccine candidate.

Merozoite surface protein 1 (MSP1) is the major protein component on the surface of the merozoite, the erythrocyte-invasive form of the malaria parasite Plasmodium. Present in all species of Plasmodium, it undergoes two distinct proteolytic maturation steps during the course of merozoite development that are essential for invasion of the erythrocyte. Antibodies specific for the C-terminal maturation product, MSP1-19, can inhibit erythrocyte invasion and parasite growth. This polypeptide is therefore considered to be one of the more promising malaria vaccine candidates. We describe here the crystal structure of recombinant MSP1-19 from P.falciparum (PfMSP1-19), the most virulent species of the parasite in humans, as a complex with the Fab fragment of the monoclonal antibody G17.12. This antibody recognises a discontinuous epitope comprising 13 residues on the first epidermal growth factor (EGF)-like domain of PfMSP1-19. Although G17.12 was raised against the recombinant antigen expressed in an insect cell/baculovirus system, it binds uniformly to the surface of merozoites from the late schizont stage, showing that the cognate epitope is exposed on the naturally occurring MSP1 polypeptide complex. Although the epitope includes residues that have been mapped to regions recognised by invasion-inhibiting antibodies studied by other workers, G17.12 does not inhibit erythrocyte invasion or MSP1 processing.

Development of a bispecific monoclonal antibody for use in molecular hybridisation.

A mouse hybrid hybridoma (tetradoma) was prepared by fusing hybridomas producing monoclonal antibody to acetyl-aminofluorene with hybridomas producing antibody against calf intestine alkaline phosphatase. The tetradoma line established secreted immunoglobulin manifesting parental and bispecific binding characteristics. Bispecific monoclonal antibody was purified and used for a one-step immunodetection assay of non-radioactive DNA and RNA probes. The immunoassay developed was able to detect 5 pg DNA within 2 h and gave low background noise.

Comparison of various methods and reagents for species identification of Staphylococcus aureus positive or negative for the mecA gene.

The reliability of various methods for species identification of Staphylococcus aureus was evaluated. A total of 135 coagulase-positive (SA) or -negative (SS) staphylococcal isolates were tested, including methicillin-resistant (MR) and -susceptible (MS) strains. When the nuc gene which encodes the S. aureus thermonuclease (TNase) was amplified in a multiplex PCR simultaneously with the mecA gene which encodes for the MR-associated penicillin-binding protein 2a of staphylococci, the nuc amplification showed full agreement with the results of the coagulase test. TNase detected by an enzymatic method or as protein in a sandwich ELISA identified S. aureus with nearly the same precision as the PCR. The Staphylase, Monostaph and Staphaurex agglutination kits were all reliable for identification of MSSA, but not for MRSA. Most of the negative MRSA strains were identified by the Pastorex agglutination kit, in which reagents for fibrinogen receptor and protein A detection have been supplemented with antibodies for capsular polysaccharides of the serotypes 5 and 8. These results show that detection of the nuc gene or its TNase product is highly reliable for identification of both MRSA and MSSA strains, while various widely used agglutination kits do not show the same reliability for identification of MRSA strains.

7 alpha-hydroxy-dehydroepiandrosterone and immune response.

In human and murine lymphoid organs, circulating 3 beta-hydroxysteroids, including pregnenolone (PREG), dehydroepiandrosterone (DHEA), and epiandrosterone (EPIA), are 7 alpha-hydroxylated by a cytochrome P450 identified in the hippocampus as P4507B1. Mouse and human lymphoid organs produced different patterns of 3 beta-hydroxysteroid 7 alpha-hydroxylation with the absence of pregnenolone and epiandrosterone hydroxylation in human and mouse, respectively. Both 7 alpha-hydroxy-DHEA and 7 alpha-hydroxy-EPIA triggered a significant increase of antitetanus toxoid and anti-Bordetella pertussis toxins IgGs production in cultures of activated B + T cells derived from human tonsils, whereas both 7 alpha-hydroxy-PREG and 7 alpha-hydroxy-DHEA increased the immune response in mouse. Paracrine action of 7 alpha-hydroxysteroids resulted from their production in cells of the lymphoid organs. Comparison of P4507B1 sequences in rat, human, and two mouse species showed that one amino acid change might explain important differences in KM for 7 alpha-hydroxylation, and suggested that such differences might contribute to the extent of immune response.

Effect of UVA-activated riboflavin on dentin bonding.

Recent studies have reported collagen cross-linking after exposure to riboflavin followed by ultraviolet-A (UVA) exposure. This study is the first to investigate the effect of a riboflavin-containing primer on adhesive interface stability and dentinal matrix metalloproteinase activity. Human dentin was etched with 35% phosphoric acid, treated with 0.1% riboflavin, exposed to UVA for 2 min, and bonded with a two-step etch-and-rinse adhesive. Adhesive was applied to control specimens without riboflavin/UVA. Specimens were subjected to microtensile bond strength tests and pulled to failure after storage for 24 hrs, 6 mos, or 1 yr. Interfacial nanoleakage was evaluated by light and transmission electron microscopy. To investigate dentinal matrix metalloproteinase activity, we performed correlative zymographic assays on protein extracts obtained from phosphoric-acid-etched dentin powder with or without riboflavin/UVA treatment and XP Bond. Ultraviolet-activated riboflavin treatment increased the immediate bond strength to dentin at all aging intervals (p < 0.05 vs. control) and decreased interfacial nanoleakage in aged specimens (1 yr; p < 0.05). Zymograms revealed that riboflavin/UVA pre-treatment inhibited dentinal matrix metalloproteinase activity (especially MMP-9). In conclusion, dentinal collagen cross-linking induced by riboflavin/UVA increased immediate bond strength, stabilized the adhesive interface, and inhibited dentin matrix metalloproteinases, thereby increasing the durability of resin-dentin bonds.

Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix.

Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule. Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated.

Purification and characterization of a major cell surface glycoprotein in Zajdela ascites hepatoma cells which displays a potent concanavalin A receptor activity.

A major cell surface sialoglycoprotein with Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells. The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation and NaIO4 followed by reduction with NaB3H4. Surface-labeled glycoproteins were released by short incubations with TPCK-trypsin at 37 degrees C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP II2, was then purified by chromatography on DEAE-cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS-polyacrylamide gels, isoelectric focusing, rechromatography on DEAE-cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,000 daltons. The location of GP II2 on the cell surface was confirmed by the fact that it could be labeled metabolically with D-(3H) glucosamine and externally through the nonpenetrating periodate-NaB3H4 system. GP II2 could not be removed from the cell surface by high salt concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP II2 could be an integral protein. Analysis of the carbohydrate composition of GP II2 revealed galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both O- and N-linked to the polypeptide chain, most of them being O-linked. Finally, GP II2 has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.

Immunological screening of a glycoprotein antigen expressed by Zajdela ascites hepatoma cells on normal rat tissues and tumour cells.

Expression of the glycoprotein MII2 antigen originally identified in Zajdela ascites hepatoma cells was investigated in several normal rat tissues and in more or less differentiated tumours using biochemical and immunological approaches. SDS-polyacrylamide gel electrophoresis followed by fluorography or immunoblotting with an antiserum raised against the purified MII2 antigen revealed that this antigen was absent from normal liver cells. ELISA assays, indirect immunofluorescence and immunoprecipitation experiments using the same antiserum showed that this glycoprotein was not expressed in various normal tissues such as liver, spleen, lung, pancreas, intestine and stomach, but it was unexpectedly detected in kidney and thymic tissues. However, the molecular weight of the antigens immunoprecipitated from kidney and thymus was lower than the one of MII2 (Mr of 60,000 versus 110,000-160,000 for purified MII2). No staining was observed in embryonic rat liver at 10 and 20 days of development. Moreover, this antigen was present on the surface of Morris hepatoma 7777, another rapidly proliferating and poorly differentiated hepatocellular carcinoma. In contrast, this antigen was not detected on the surface of in vitro Zajdela hepatoma cells (ZHC) or of partially differentiated hepatomas (Faza) which have recovered some hepatic functions. In addition, the MII2 antigen was found on the human non-hepatic HT-29 tumour cell line, under its undifferentiated form (HT-29 G+ subline). The possible relationships between the expression of this antigen and both the malignant transformation process and the differentiation process are discussed.

Similar binding properties for a neutralizing anti-tetanus toxoid human monoclonal antibody and its bacterially expressed Fab.

A high-affinity anti-tenanus toxoid (TT) human monoclonal antibody showing neutralizing activity was isolated from a fusion between mouse myeloma and human splenic cells. Fab fragments from this antibody were obtained using a recombinant phage surface-display expression system. The parental antibody and the corresponding Fab had identical immunological activities, including specificity and affinity. These results confirm the feasibility of developing Escherichia coli expression of monoclonal human Fab from hybridoma cells.

Interactions of HasA, a bacterial haemophore, with haemoglobin and with its outer membrane receptor HasR.

The major mechanism by which bacteria acquire free or haemoglobin-bound haem involves direct binding of haem to specific outer membrane receptors. Serratia marcescens and Pseudomonas aeruginosa have an alternative system, which involves an extracellular haemophore, HasA, that captures free or haemoglobin-bound haem and shuttles it to a specific cell surface outer membrane receptor, HasR. Both haem-free (apoprotein) and haem-loaded (holoprotein) HasA bind to HasR, evidence for direct protein-protein interactions between HasA and HasR. HasA binding to HasR takes place in a tonB mutant. TonB is thus required for a step subsequent to HasA binding.