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M Pintor

Publications and source records attributed to M Pintor.

14 recordsLinked to original sources
PubMed1998

Analysis of TbpA and TbpB functionality in defective mutants of Neisseria meningitidis.

Iron uptake analysis suggested that the Neisseria meningitidis transferrin (Tf) binding proteins, TbpA and TbpB, form only one type of receptor complex. Mutants defective in the synthesis of either TbpA or TbpB, but not defective in both proteins, can bind Tf, suggesting that both proteins are surface exposed and function in Tf binding. Also, iron uptake from Tf into the meningococci did not require the presence of both Tbps. The TbpB-defective mutant incorporated c. 37% of the iron taken up by the wild-type strain, but this was insufficient for bacterial growth. The TbpA-defective mutant incorporated c. 50% of the iron taken up by the wild-type strain and was able to grow with Tf as the only iron source. Mouse antibodies specific for TbpA were able to block c. 70% of the iron uptake from Tf in the wild-type strain, whereas they blocked only 22% of iron uptake in the TbpB-defective mutant and did not block uptake in the TbpA-defective strain. These results emphasise that TbpA should be considered in future vaccine trials in which iron-restricted proteins are to be included in the vaccine formulation.

Antibodies, Blocking↗
PubMed1996

Blocking of iron uptake from transferrin by antibodies against the transferrin binding proteins in Neisseria meningitidis.

Neisseria meningitidis, when cultured in iron-restricted environments, synthesises new outer-membrane proteins, many of which are necessary for their survival and growth. Some of these proteins e.g. transferrin-binding proteins 1 and 2 (Tbp1 and Tbp2), are required for the acquisition of iron from transferrin and are examples of important iron-regulated meningococcal surface antigens which are not expressed after growth in common laboratory media. The antigenicity and antigenic heterogeneity of these proteins have been extensively studied, and the bactericidal activity of antibodies directed to them have been studied. In this work we analysed the ability of such antibodies to inhibit transferrin binding and to block iron uptake from human transferrin. Antisera from mice immunized with either meningococcal outer membrane vesicles, purified Tbp1/2 complexes, or purified Tbp2, were incorporated in radiolabeled-iron uptake assays. Uptake was blocked by more than 80% in the homologous strains, but blocked much less efficiently in some heterologous strains, correlating well with inhibition of transferrin binding and with an inhibitory effect on bacterial growth. Inhibition of iron uptake from citrate was unaffected which suggests that this effect is due to antibodies against the components of the transferrin binding system, specially Tbp2. Our results support the importance of these proteins and their suitability to be considered in the development of effective vaccines against serogroup B meningococci.

Antibodies, Bacterial↗
PubMed1996

Antigenicity, cross-reactivity and surface exposure of the Neisseria meningitidis 37 kDa protein (Fbp).

The 37 kDa iron-repressible protein, Fbp, was purified from two Neisseria meningitidis strains by metal-affinity chromatography and used to obtain mouse monospecific polyclonal immune sera. Dot-blot, immunoblotting and whole cell ELISA results demonstrate that the Fbp is present in all 16 N. meningitidis and four commensal Neisseria species tested, is highly antigenic in mouse when injected in pure form, and shows intra- and inter-species antigenic homogeneity, anti-Fbp antibodies being fully cross-reactive using the techniques mentioned. We also found that Fbp molecules (or parts of them) are surface exposed, in disagreement with the proposed exclusively periplasmic localization, although anti-Fbp antibodies seem unable to block iron uptake or to induce complement-mediated killing of the meningococci. Taken along with the high immunogenicity of the purified protein and the complete cross-reactivity of the antibodies elicited, this suggests that the protective effect of the purified Fbp must be further studied to evaluate its inclusion in future vaccine trials.

Animals↗
PubMed1996

Blocking of iron uptake by monoclonal antibodies specific for the Neisseria meningitidis transferrin-binding protein 2.

The existence of epitopes common to different strains in the Neisseria meningitidis transferrin (Tf)-binding protein 2 (TBP2), combined with the ability of polyclonal anti-TBP2 antibodies to inhibit Tf binding and block iron uptake in this species, led to this study on the effect of anti-TBP1+2 monoclonal antibodies (MAbs) to determine the presence of epitopes inside the Tf-binding region. All MAbs used reacted exclusively with the homologous strain when tested by dot-blots of outer membrane vesicles, with the reaction being specific for TBP2 after SDS-PAGE and electroblotting. In contrast, ELISA and iron-uptake blocking assays were also positive with heterologous strains belonging to Rokbi's group II (high mol.wt TBP2). The results confirmed the two group classification proposed by Rokbi and, in contrast to other studies, indicated the existence of epitopes in the Tf-binding region that are common only to strains of Rokbi's group II. These epitopes may become denatured after drying for dot-blot assays or after SDS-PAGE and electroblotting.

Animals↗
PubMed1994

Reliability of laboratory models in the analysis of TBP2 and other meningococcal antigens.

The lack of experimental models suitable for the study of meningococcal pathogenicity led us to investigate if those actually in use (culture in iron-restricted media and animal models) provide results comparable with the responses observed in vivo during infection. In this work we studied three invasive strains cultured both in laboratory media and in human plasma, analysing the immune responses elicited in mice against membrane antigens and comparing them with those seen using homologous human convalescent sera. Outer membrane protein profiles observed after culture in plasma were different and more complex than those obtained after growth in laboratory media. Analogous differences were observed in the antigenic profiles, detecting some antigens recognized by human, but not mouse sera, and vice versa. However, the response to one of the major iron-regulated outer membrane antigens, the transferrin binding protein 2 (TBP2), was unaffected by the culture medium or the model, human or mouse, used for the analysis. In conclusion, we have found that results of antigenic analysis change depending on the culture conditions and animal models used. For the meningococcal antigen TBP2, growth in iron-restricted laboratory media and a mouse model provide results which correlate well with those observed using convalescent human serum from individuals recovered from infections. We suggest that careful analysis and evaluation of experimental results and their comparison with in vivo elicited immune responses are essential in order to get accurate extrapolations for experimental vaccine designs.

Animals↗
PubMed1993

Characterization of the transferrin-iron uptake system in Neisseria meningitidis.

The transferrin-iron uptake system of six Neisseria meningitidis strains was characterized using 125I-transferrin in receptor assays and 55Fe-loaded transferrin in uptake assays. Receptors for transferrin varied among the strains both in number (from 700 to 4700 receptors per cell) and in their affinity constants for the protein (Ka ranged from 0.7 x 10(7) to 4.0 x 10(7) l mol-1). Neither receptor numbers nor affinity constants were significantly different in carrier and invasive strains, although the Ka seem to be somewhat higher in the latter. Iron uptake from transferrin was also variable among the strains, but showed the same lack of correlation with their origin.

Biological Transport, Active↗
PubMed1992

Immunogenicity and antigenic heterogeneity of a human transferrin-binding protein in Neisseria meningitidis.

Growing Neisseria meningitidis on an iron restriction medium induces the synthesis of new outer membrane proteins, some of them true iron-regulated outer membrane proteins (IROMPs) and others synthesized because of the stress produced by the iron restriction. Some of these proteins are antigenic and can be considered for the development of vaccines; this is especially desirable in the case of N. meningitidis serogroup B, for which polysaccharide vaccines are not efficient. The antigenicity of N. meningitidis 37- and 70-kDa IROMPs has been studied previously; in this work, we studied the immunogenicity and antigenic heterogeneity of another IROMP, the human transferrin-binding protein 2 (TBP2), which seems to be indispensable for meningococcal growth inside the host. Mice were inoculated with purified outer membrane vesicles (blebs) from 5 selected N. meningitidis strains, and the five serum samples obtained were analyzed for anti-TBP2 antibodies by using the homologous strain and for cross-reactivity with the TBP2 of the 4 other selected strains and another 35 heterologous N. meningitidis strains. The TBP2s of the 5 strains tested were all immunogenic in mice to various degrees depending on the strain, and all five TBP2s shared one or more epitopes with heterologous strains (as shown by the cross-reactivities of the five serum samples), although the number of cross-reacting strains was very variable, ranging from 2 for strain V002 to 35 for strain P391. This suggests that the TBP2 epitopes of different strains differ in nature or in their accessibility to the immune system. Under the iron restriction conditions used, all strains synthesized a non-TBP2 antigenic 56-kDa protein thought to be a stress protein.

Animals↗
PubMed1991

Analysis of the molecular mass heterogeneity of the transferrin receptor in Neisseria meningitidis and commensal Neisseria.

Peroxidase-conjugated transferrin was used to detect transferrin receptors both in intact outer membrane vesicles (OMVs) from Neisseria species in a dot blot assay, and in SDS-PAGE-separated OMV proteins after transferring to nitrocellulose membranes. All N. meningitidis strains produced transferrin receptors after culturing in either iron sufficiency or iron restriction although expression was higher in the latter case, whereas only six N. lactamica and two N. sicca (among 20 commensal species) were able to bind transferrin. Molecular mass (MM) of the receptors were mainly between 78 kDa and 85 kDa (87.5% of strains), 12.5% had receptors with MM close to 70 kDa, and 5% showed receptors with MM over 85 kDa. Our results confirm the molecular mass heterogeneity of the transferrin receptors in N. meningitidis, completely disagree with the 'universal' 98 kDa receptor proposed by some authors, and show a low expression of the receptor in commensal Neisseria.

Densitometry↗
PubMed1990

Analysis of the expression of outer-membrane proteins in Neisseria meningitidis in iron-replete and iron-deficient media.

A total of 103 Neisseria strains, including 42 carrier and 40 invasive N. meningitidis and 21 commensal species were studied. Outer-membrane proteins from carrier and invasive N. meningitidis showed similar molecular mass distributions (except in the range from 78 to 82 kDa in which 90% of the invasive but only 47.6% of the carrier strains expressed proteins), many strains showing neither class II (41 kDa) nor class III (38 kDa) proteins. When grown in iron-restricted conditions proteins were induced mainly in the ranges from 62 to 92 kDa with no significant differences between groups. Commensal species, both in normal and in iron-restricted conditions, showed outer-membrane protein patterns different from those of N. meningitidis in several molecular mass ranges. Cluster analysis based on expression of principal outer-membrane proteins allowed the differentiation of commensal species and N. meningitidis, although not that of the invasive and carrier groups within the latter.

Bacterial Outer Membrane Proteins↗
PubMed1990

Levels of biogenic amines, their metabolites, and tyrosine hydroxylase activity in the human epileptic temporal cortex.

The levels of serotonin (5-HT), 5 hydroxyindoleacetic acid (5-HIAA), dopamine (DA), homovanillic acid (HVA), norepinephrine (NE), and tyrosine hydroxylase (TH) activity were measured in the focus (spiking) and nonfocus (nonspiking) regions of the temporal neocortex of 20 patients with intractable complex partial seizures. The levels of 5-HT, DA, 5-HIAA, and HVA were higher in the focus when compared to the nonfocus. Values for NE and TH activity were not different when focus and nonfocus were compared. The ratios of metabolite to precursor for 5-HT and DA were not significantly different between the focus and the nonfocus, suggesting that the changes observed were the result of a modification in the synthesis and release of these amines. Such changes in the epileptic focus could be caused by altered transsynaptic regulatory processes, which occur as a result of neuronal loss, gliosis, or neuronal sprouting.

Adolescent↗
PubMed1990

Iron and outer membrane proteins in the susceptibility of Neisseria meningitidis to human serum.

The proportion of carrier-isolated Neisseria meningitidis strains sensitive to human serum (37.2%) was found to be significantly higher than that of case-isolated ones (4.1%), although the difference is too low to consider serum-resistance responsible for invasion in this microorganism. Serum-susceptibility was not related to the existence of specific outer membrane proteins, as is the case of N. gonorrhoeae. Iron restriction induced iron-regulated outer membrane proteins in each strain (but not the same proteins in all strains) but without any detectable effect on serum-susceptibility. Iron excess was also unable to induce changes in the susceptibility of N. meningitidis to human serum.

Bacterial Outer Membrane Proteins↗