Biomedical subjects

M E Patarroyo

Publications and source records attributed to M E Patarroyo.

At least 19 recordsLinked to original sources

PubMed2006

Comparative molecular and three-dimensional analysis of the peptide-MHC II binding region in both human and Aotus MHC-DRB molecules confirms their usefulness in antimalarial vaccine development.

A vaccine against malaria is desperately needed, and Aotus monkeys are highly susceptible to experimental infection with malarial parasites. A thorough analysis of this monkey's immune system molecules was thus undertaken in our institute. Cloning and sequencing, followed by three-dimensional analysis, has revealed high homology with some HLA-DRB1 molecules in terms of their peptide binding region pockets. Molecules such as HLA-DRB1*03, 11, 08, and HLA-DRB1*04 are so similar to Aotus MHC-DRB molecules that peptides identified as binding to these molecules and inducing protective immunity in these monkeys could be used in humans without further refinement, while small modifications seem to be needed for those binding to HLA-DRB1*07, HLA-DRB1*15, 16, and HLA-DRB1*10-like molecules, making this New World monkey an excellent model for tailor-made vaccine development, especially against malaria.

Amino Acid Sequence↗

PubMed2006

Synthetic peptides from Plasmodium falciparum apical membrane antigen 1 (AMA-1) specifically interacting with human hepatocytes.

Plasmodium falciparum apical membrane antigen 1 (AMA-1) is expressed during both the sporozoite and merozoite stage of the parasite's life cycle. The role placed by AMA-1 during sporozoite invasion of hepatocytes has not been made sufficiently clear to date. Identifying the sequences involved in binding to hepatocytes is an important step towards understanding the structural basis for sporozoite-hepatocyte interaction. Binding assays between P. falciparum AMA-1 peptides and HepG2 cell were performed in this study to identify possible AMA-1 functional regions. Four AMA-1 high activity binding peptides (HABPs) bound specifically to hepatocytes: 4310 ((74)QHAYPIDHEGAEPAPQEQNL(93)), 4316 ((194)TLDEMRHFYKDNKYVKNLDE(213)), 4321 ((294)VVDNWEKVCPRKNLQNAKFGY(313)) and 4332 ((514)AEVTSNNEVVVKEEYKDEYA(533)). Their binding to these cells became saturable and resistant to treatment with neuraminidase. Most of these peptides were located in AMA-1 domains I and III, these being target regions for protective antibody responses. These peptides interacted with 36 and 58 kDa proteins on the erythrocyte surface. Some of the peptides were found in exposed regions of the AMA-1 protein, thereby facilitating their interaction with host cells. It is thus probable that AMA-1 regions defined by the four peptides mentioned above are involved in sporozoite-hepatocyte interaction.

Amino Acid Sequence↗

PubMed2005

Identification of five different IGHV gene families in owl monkeys (Aotus nancymaae).

In order to characterize immunoglobulin heavy-chain variable (IGHV) genes in Aotus nancymaae monkeys, different mRNAs encoded by five IGHV families in this non-human primate were molecularly analysed considering their paramount importance in antibody production in an immune response. This study reports gene products exhibiting 91% amino acid similarity with IGHV1, IGHV2, IGHV3, IGHV4 and IGHV7 human IGHV families. Our analyses suggest that the IGHV gene has several conserved characteristics in humans and A. nancymaae. Several amino acid residues that are highly conserved in all family members described in humans were also present in these families in A. nancymaae. Antibody diversity in these families has remained the same since divergence of both species. Our study continues to provide evidence supporting the use of A. nancymaae monkey as an animal model for studying antibody response.

Amino Acid Sequence↗

PubMed2005

Based on HLA-DR beta1* allele binding specificities, striking differences in distance and TCR Contacting Residue Orientation can be observed in modified protection-inducing malarial synthetic peptides.

An anti-malarial vaccine is urgently needed, especially against P. falciparum which causes 2 to 3 million deaths each year, mostly in Sub-Saharan African children. This vaccine should contain molecules from the parasite's different developmental stages due to the parasite's remarkable complexity and genetic variability. The first approach using synthetic peptides from different parasite stage molecules (the SPf66 malaria vaccine) conferred limited protective efficacy in Aotus monkeys and in large field-trials carried out in different parts of the world SPf66 contains red blood cell (RBC) binding merozoite peptides for which immune responses against them are genetically controlled by HLA-DR region. Therefore, a systematic search of conserved high activity binding peptides (HABP) was undertaken aimed at using them as immunogens. However, these peptides were poorly immunogenic and had poor protection-inducing capacity against experimental challenge with a P. falciparum strain highly infective for Aotus monkeys an experimental model with an immune system quite similar to humans. Modifications were thus made to key residues to render them immunogenic and protection-inducing. These native and modified HABPs' three-dimensional structure was determined by (1)H-NMR studies and their ability in forming stable Major Histocompatibility Class II - peptide (MHCII-peptide) complexes was correlated with their ability to bind in vitro to purified HLA-DR beta1* molecules. Our experimental data suggests a correlation between modified HABPs' three-dimensional structure, HLA-DR beta1* binding preferences and their protection-inducing capacity in monkeys. Furthermore, the data presented here indicates that a synthetic peptide vaccine's three-dimensional structural features dictate both HLA-DR beta1* allele binding preference (imposing genetic restriction on the immune response) and on these vaccines' protection-inducing value. Basic knowledge of a parasite's functionally active peptides, their 3D structure and their interaction for forming the MHC II- peptide-TCR complex will thus contribute towards designing fully effective multi-component, multi-stage subunit-based malarial vaccines.

Alleles↗

PubMed2004

Potent, long lasting systemic antibody levels and mixed Th1/Th2 immune response after nasal immunization with malaria antigen loaded PLGA microparticles.

The immunogenicity of the synthetic malaria vaccine SPf66 has been recently improved by the application of new adjuvants as QS-21 saponin or poly-D,L-lactide-co-glycolide (PLGA) polymers. The search for less invasive administration routes made us test the immunogenicity of SPf66-loaded microparticles by the nasal route in Balb/c mice. We report here that the intranasal administration of the adequate PLGA vaccine formulations greatly improves and maintains higher antibody levels compared to the conventional alum adjuvant and to the administration of the particles by other routes (subcutaneous, oral). Systemic immune responses were characterized as mixed Th1/Th2-type: IFN-gamma and IgG2a isotype were found as signs of Th1 activation, whilst IgE and IgG1 secretions indicate Th2 response. Since both types of response have been associated to protective immunity in malaria, we postulate that this new approach supposes an advantage over the traditional adjuvants and routes.

Administration, Intranasal↗

PubMed2004

Isolation and identification of mycobacteria in New World primates maintained in captivity.

The presence of several Mycobacterium species was determined in 68 New World monkeys kept captive in the Cali Zoo. One hundred and thirty-three gastric lavage and blood samples were evaluated for mycobacterial presence by Ziehl-Neelsen (ZN) staining, culture and PCR amplification of the Mycobacterium tuberculosis Mtp40 species-specific gene. Mycobacteria other than tuberculosis (MOTT) were identified by PCR restriction fragment length polymorphism (RFLP). Different species of mycobacteria were detected in 65% of the primate population studied by Alpha Antigen PCR. Eleven percent were positive for Mtp40 PCR amplification, being diagnosed as having M. tuberculosis, and acid-fast bacilli were observed in 23% by ZN staining. MOTT were isolated from samples taken from 37 primates by culturing; according to the RFLP analysis, three strains were classified as belonging to the MAISS complex (Mycobacterium avium-intracellulare-scrofulaceum-simiae) and eight more, isolated from soil inside the cages, were categorized as environmental contaminants. Mycobacterium spp. were detected in 13 different New World primate species showing that PCR amplification of the Mtp40 gene is a better tool than culture for M. tuberculosis detection in captive animals and that RFLP is a useful technique for MOTT identification.

Animals↗

PubMed2004

Stability indicating method for SPf66 antimalarial peptide in solution.

Stability studies on the SPf66 antimalarial peptide with different pH and temperature conditions were carried out. The degradation mechanism was elucidated by the size-exclusion chromatography (SEC) technique and the experimental data obtained at 37 degrees C and different pH were fitted to a kinetic degradation model that could explain the loss of its immunogenic capacity. At 5, 25, 37, and 70 degrees C and pH 2, changes were detected in the areas of the different species, although the values obtained could not be fitted to any known degradation kinetics.

Chromatography, Gel↗

PubMed2004

Comparative analysis of CD45 proteins in primate context: owl monkeys vs humans.

Transmembrane protein tyrosine phosphatase (PTPase) CD45 has been implicated in activating, differentiating and the development of different immune system cells. It regulates T-or B-cell activation during receptor-specific recognition by dephosphorylating tyrosine residues in protein kinase substrates. Aotus nancymaae, Aotus nigriceps, and Aotus vociferans CD45 nucleotide and deduced amino acid sequences are presented here, where we found 90-92% identity with the human counterpart in the nucleotide sequence and 83-86% in the amino acid sequence. Aotus CD45 alternative splicing isoforms include the same exons used in human CD45, producing several identical molecular weight nucleotide fragments. Most of the non-synonymous substitutions were found in the extracellular domain. The more conserved CD45 cytoplasmic portion has two intracellular phosphatase domains (D1 and D2) separated by a short spacer and some residues and motifs involved in signaling or molecular docking, intra- and intermolecular interactions and CD45 activity and activity regulation. All invariant residues and structural/functional motifs found in PTPases were totally conserved, suggesting that Aotus CD45 is a functional enzyme. Phylogenetic analysis has shown that the Aotus CD45 molecules are more related to the human homologs than to other reported vertebrate sequences and that the ancestral group of Aotus clade is A. vociferans. When Aotus species were compared, A. nigriceps and A. vociferans were the two most distant species, while A. nancymaae and A. nigriceps appeared to be a sister group. This could be relevant in deciding which Aotus species is to be used for studying particular immunological processes during lymphocyte activation or development.

Alternative Splicing↗

PubMed2003

Immune response after oral administration of the encapsulated malaria synthetic peptide SPf66.

The synthetic peptide SPf66 adsorbed on alum is one of the few Plasmodium falciparum vaccines which have been tested in field trials. We previously reported that subcutaneous administration of SPf66 loaded PLGA microparticles (MP) enhances the antibody response to this antigen compared to the conventional alum formulation. We now evaluate the suitability of polymeric formulations to obtain systemic immune responses by gastric intubation of Balb/c mice. Formulations composed of 1:1 mixtures of PLGA 50:50 and 75:25 (lactic:glycolic) microparticles were administered by the oral route, and when animals were boosted 3 weeks later significant systemic IgG antibody responses were elicited, comparable to alum triple shot and superior to the aqueous vaccine given by the oral route. The finding of IgG2a isotype for PLGA-vaccinated mice compared to the absent levels of this isotype for the alum-vaccinated group could be interpreted as a sign of Th1-like immune response and cellular immune response activation. Our results confirm that using the appropriate schedule the oral administration of PLGA particles is suitable to obtain systemic immune responses to the carried antigen.

Adjuvants, Immunologic↗

PubMed2003

Alpha helix shortening in 1522 MSP-1 conserved peptide analogs is associated with immunogenicity and protection against P. falciparum malaria.

1522 is a nonimmunogenic conserved high-activity binding peptide (HABP) belonging to Plasmodium falciparum MSP-1 protein N-terminal fragment. The key amino acids in binding to red blood cells (RBC) were identified and replaced by others having similar mass but different charge. Because conserved HABPs are not antigenic nor immunogenic, immunogenicity and protectivity studies were then conducted on them in the Aotus monkey. 1H-NMR studies included the lead peptide 1522 as well as the analogs 9782, 13446, 13448, and 13442 to relate their structure to biological function. All the peptides presented alpha-helical structure, with differences observed in helix location and extension. The nonprotective 1522 peptide was totally helical from the N- to the C-terminus, very similar to nonprotective 13442 and 13448 peptides whose extension was almost totally helical. The 9782 and 13446 protective peptides, however, possessed a shorter helical region where modified critical binding residues were not included. A more flexible region was generated at the C-terminus in those peptides with a shorter helical region, leading to a greater number of conformers. These data suggest that peptide flexibility results in increased interaction with immune system molecules, generating protective immunity.

Amino Acid Sequence↗

PubMed2003

Alpha1 and alpha2 domains of Aotus MHC class I and Catarrhini MHC class Ia share similar characteristics.

Functional and structural analyses of major histocompatibility complex (MHC) class I molecules of the Aotus genus are necessary to validate it as a solid animal model for biomedical research. We thus isolated, cloned and sequenced exons 2 and 3 from three Aotus species (A. nancymaae, A. nigriceps and A. vociferans). We found 24 sequences, which divided into two different groups (Ao-g1 and Ao-g2). A further sequence was identified as a processed pseudogene (Aona-PS2). Both sequence evolution and variability analyses showed that Ao-g1 and Ao-g2 display similar characteristics to Catarrhini's classical loci, such as positive selection pressure at the peptide binding region (PBR) high variability and a trans-specific evolution pattern.

Amino Acid Sequence↗

PubMed2003

Plasmodium falciparum EBA-140 kDa protein peptides that bind to human red blood cells.

The erythrocyte-binding antigen 140 (EBA140) sequence was chemically synthesized in 61 20-mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte-binding assays. Peptides 26135, 26144, 26147, 26160, 26170 and 26177 presented high erythrocyte-binding activity, with affinity constants ranging from 350 to 750 nM. Critical erythrocyte-binding residues were determined by competition-binding assays with glycine analogous peptides. Cross-linking assays with SDS-PAGE from high erythrocyte membrane protein binding peptides showed that all these peptides bound specifically to 25, 52 and 75 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide-binding assays using enzyme-treated erythrocytes, showing that these protein receptors are susceptible to structural changes provoked by enzyme treatment (neuraminidase, trypsin or chymotrypsin). Inhibition invasion assays in 'in vitro' cultures showed that all specific high binding sequences were able to inhibit invasion by 11-69% at 200 microM concentration.

Amino Acid Sequence↗

PubMed2003

Characterizing T-cell receptor gamma-variable gene in Aotus nancymaae owl monkey peripheral blood.

Gammadelta T lymphocytes have a heterodimeric complex formed by the association of gamma and delta chains as receptor. Proliferation of this lymphocyte population has been observed, when infection by several pathogens such as Mycobacterium tuberculosis and Plasmodium spp. occurs. The New World Monkey Aotus nancymaae has become a very good experimental model for the immunological and physiopathological study of these infectious agents. The A. nancymaae gamma-variable region was characterized from peripheral blood samples by using cDNA and genomic DNA polymerase chain reaction amplification, DNA sequencing, and dot-blot hybridization techniques. Seventeen different T-cell receptor gamma-variable (TCRGV) sequences were obtained. These sequences were distributed among TCRGV subsets 1, 2, or 3, according to human subset classification. Although no subset 4 amplification was obtained, this subset was detected by dot-blot hybridization. The presence of these 4 subsets resembles the behavior displayed by 'gammadelta-low species' (humans and mice), where high diversity among these lymphocytes can be observed. Homologies greater than 70% were found with respect to humans. Sequence convergence between human and A. nancymaae subsets 1 and 3 highlights Aotus as a promising model for studying these lymphocyte functions.

Amino Acid Sequence↗

PubMed2003

Immunodiagnosis of parasitic diseases with synthetic peptides.

Parasitic diseases remain as a major public health problem worldwide, not only based on their historically high morbidity and mortality rates, but also because risk factors associated with their transmission are increasing. Laboratory diagnosis and particularly immunodiagnosis is a basic tool for the demonstration, clinical management and control of these infections. Classically, the serological tests for the detection of antibodies or antigens are based on the use of crude and purified antigens. Synthetic peptides have opened a new field and perspectives, as the source of pure epitopes and molecules for diagnosis of malaria, Chagas' disease, leishmaniasis, schistosomiasis, hidatidosis, cysticercosis and fasciolosis based on the detection of antibodies and circulating antigens. Herein, are critically reviewed the relevant advances and applications of the synthetic peptides on immunodiagnosis of parasitic diseases. A variety of sequences, constructs (monomers, polymers, MAPs), immunological methods and samples have been used, demonstrating their diagnostic potential. However, in most parasitic infections it is necessary to use more than a single peptide in order to avoid the genetic restriction against certain epitopes, as well as to test them in well characteized groups of patients, in order to confirm their sensitivity and specificity. The concept of multidiagnosis with synthetic peptides, using a novel multi-dot blot assay is introduced. Finally, the chemical imitation of antigens, offers a tremendous posibilities in the diagnosis of parasitic infections in developing countries since this strategy is cheaper, simpler, reproducible, useful for large scale testing and in most cases, specific and sensitive.

Animals↗

PubMed2002

Immunization of cattle with synthetic peptides derived from the Boophilus microplus gut protein (Bm86).

Three synthetic peptides (SBm4912, SBm7462 and SBm19733), derived from the Bm86 glycoprotein from Boophilus microplus gut, were constructed and used to immunize cattle from a tick-free area. The immunized animals received three subcutaneous doses of the peptides, with saponin as adjuvant, at 30-day intervals. The immune response was evaluated by IgG elicited against the peptides by the detection of anti-Bm86 specific antibodies in situ and by Western blotting analysis. After tick challenge, reduction in the number, weight and oviposition capacity of engorged females was observed in the tick population that had fed on immunized animals. The results pointed a high efficacy (81.05%) for the SBm7462 synthetic peptide in relation to the others (p<0.01), demonstrating the efficiency of the immune response elicited by synthetic peptides to control the cattle tick B. microplus.

Animals↗

PubMed2002

Remarkably high antibody levels and protection against P. falciparum malaria in Aotus monkeys after a single immunisation of SPf66 encapsulated in PLGA microspheres.

Single dose immunisation is a major goal in vaccine design. The purpose of this study was the development of a single dose delivery system for the SPf66 malaria vaccine, based on this antigen's microencapsulation in PLGA microspheres by double emulsion method. Results indicate that a single immunisation in mice and monkeys with the SPf66 malaria vaccine, encapsulated in a mixture of two formulations of PLGA microspheres, induced a remarkably high and long-lasting immune response as assessed by ELISA and Western Blott. This immune response was associated with a good protective capacity in Aotus monkeys, after experimental challenge, indicating that antigen integrity lasted following the microencapsulation process. PLGA biodegradable microspheres thus serve as an effective delivery system for the design of a single dose immunisation vaccine, such as the SPf66 synthetic malaria vaccine.

Alum Compounds↗

PubMed2002

Double dimer peptide constructs are immunogenic and protective against Plasmodium falciparum in the experimental Aotus monkey model.

Multiple antigen peptide constructs (MAPs) have been used to obtain defined multimeric peptide molecules useful in the development of possible synthetic malaria vaccines. In this context, a method was developed, named double dimer constructs (DDCs), involving the direct synthesis of a dimeric peptide with a C-terminal cysteine. A tetrameric molecule was then obtained by oxidation of sulfhydryl groups. Dimer synthesis was optimized using a Fmoc/tBu strategy, dimers were purified by HPLC, oxidized with DMSO and characterized by HPLC and MALDI-TOF-MS. The tetramers or DDCs obtained by this method were used as immunogens in the search for a possible malaria vaccine. It was found that they were immunogenic in the experimental Aotus monkey model, and were able to induce protective immunity when challenged experimentally with a highly infective Plasmodium falciparum malaria strain.

Amino Acid Sequence↗

PubMed2001

Functional and structural similarity of V gamma 9V delta 2 T cells in humans and Aotus monkeys, a primate infection model for Plasmodium falciparum malaria.

Gammadelta T cells are implicated to play crucial roles during early immune responses to pathogens. A subset of human gammadelta T cells carrying the Vgamma9Vdelta2 TCR recognize small, phosphorylated nonpeptidic Ags. However, the precise role of these cells and the ligands recognized in human immune responses against pathogens remains unclear because of the lack of suitable animal models. We have analyzed the reactivity of spleen cells of the New World monkey Aotus nancymaae against isopentenyl pyrophosphate (IPP), a phosphorylated microbial metabolite selectively activating Vgamma9Vdelta2 T cells. Spleen cells were stimulated by IPP and the expanding cell population expressed the Vgamma9 TCR. TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated cells of Aotus were analyzed by RT-PCR and DNA sequencing. The TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated Aotus and human gammadelta T cells were similar with respect to 1) TCR gene segment usage, 2) a high degree of germline sequence homology of the TCR gene segments used, and 3) the diversity of the CDR3 regions. Phylogenetic analysis of human, Pan troglodytes, and A. nancymaae TRGV gene segments showed that the interspecies differences are smaller than the intraspecies differences with TRGV9 gene segments located on a distinct clade of the phylogenetic tree. The structural and functional conservation of Vgamma9Vdelta2 T cells in A. nancymaae and humans implicates a functionally important and evolutionary conserved mechanism of recognition of phosphorylated microbial metabolites.

Amino Acid Sequence↗