PubMed Health⌕ Search

Biomedical subjects

M E Patarroyo

Publications and source records attributed to M E Patarroyo.

At least 73 records · Page 4Linked to original sources

Malaria vaccines.

Significant progress has been made in the development of the malaria vaccine during the last 20 years. Ninety percent of the 300-500 million clinical cases of malaria per year worldwide occur in Africa. Thus, research must be directed toward the 1 million African children under 5 years of age who die every year of malaria. An asexual blood-stage vaccine, capable of reducing severe and complicated malaria and malaria-related mortality, is therefore an important public health tool in these countries. Although knowledge of the parasite's biology is incomplete, research has allowed insight into some of the mechanisms that the parasite uses to evade host immunity. This is the basis for adopting an "antigenic cocktail" approach toward obtaining a synthetic or recombinant subunit vaccine such as the synthetic Colombian Malaria vaccine SPf 66. During the development of Spf66, field trials under both low and high malaria endemicity areas in Latin America and Africa have been carried out. The results from these studies showed a protective efficacy ranging between 38.8 and 60.2% against Plasmodium falciparum malaria. Given the characteristics of the normal immune response to malaria (relatively short-lived and not completely effective), it is understandable that the main goal is to try to increase the host's natural immunity. The best candidates for designing a malaria vaccine are the proteins required for parasite survival, those with low mutation rates and conserved epitopes. Because these proteins play an important role in multiple or alternative steps during the invasion process, they should be the targets against which a protective immune response should be elicited. The interaction between the malaria parasite and its host is complex. It is therefore crucial to define new ways of improving the immune response-such as directly modifying the chemical structure of epitopes or using new adjuvants or DNA immunization techniques-to produce novel vaccines against this disease.

Animals↗

Mapping of the linear antigenic determinants of the Leishmania infantum hsp70 recognized by leishmaniasis sera.

Mapping of antigenic determinants of the Leishmania infantum Hsp70 was studied by analysis of the reactivity of sera from dogs with natural visceral leishmaniasis against a collection of peptides representing overlapping sequences of the Hsp70. Despite the considerable variation in the immune response among individuals three immunodominant regions were revealed encompassing residues 241 to 260 (region I), 435 to 469 (region II) and from amino acid 601 to the carboxyl-terminal end of the protein (region II. Since anti-peptide H17 antibodies purified from a pool of leishmaniasis sera recognized the L. infantum Hsp70 and since they did not react with the homologous Hsp70s from other trypanosomatids, such as Trypanosoma cruzi, Trypanosoma rangeli and Leishmania panamensis, it was concluded that peptide H17 (region II contains an immunodominant B cell species-specific epitope. Our data indicate, however, that the complete recombinant L infantum Hsp70 protein cannot be used as a disease-specific tool for serodiagnosis since it is also recognized by the sera from patients with Chagas' disease.

Amino Acid Sequence↗

Evaluation of SPf66 malaria vaccine during a 22-month follow-up field trial in the Pacific coast of Colombia.

A double-blind randomized placebo-controlled field trial with the SPf66 malaria vaccine was carried out in an endemic area consisting of 14 small villages with exclusive fluvial access, in a rain forest area along the Rosario River, Colombia. A total of 1257 subjects completed the full three dose vaccination schedule on days 0, 30 and 180 (643 vaccinated group/623 placebo group) and were followed-up by passive and active surveillance over a period of 22 months. One hundred and thirty-four Plasmodium falciparum malaria episodes were detected (53 in vaccinated group/81 in placebo group), yielding an attack rate of 5.47 cases/100 person years of follow-up (pyears) in the vaccine group and 8.44/100 pyears in the placebo group. The estimated vaccine protective efficacy was 35.2% (95% CI 8.4-54.2%, P = 0.01). This result supports earlier findings that the SPf66 malaria vaccine diminishes the risk of infection by P. falciparum in endemic areas of South America.

Adolescent↗

Characterization of the antigenic determinants of the Leishmania infantum histone H3 recognized by antibodies elicited during canine visceral leishmaniasis.

In the present study we show that sera from dogs naturally infected with the protozoan parasite Leishmania infantum contain antibodies that specifically react with the parasite histone H3. Using synthetic peptides covering the complete sequence of the protein we located the linear antigenic determinants within the 40 amino-terminal amino acids of the molecule. In addition to the complete form of the protein (rLiH3), two regions of the Leishmania histone H3 were expressed as recombinant proteins: the rLiH3-Nt fragment containing the 39 amino-terminal amino acids and the rLiH3-Ct fragment containing the 90 carboxyl-terminal residues. Competition experiments using the protein fragment rLiH3-Nt as competitor confirmed that the antigenic determinants of histone H3 are confined to the amino-terminal domain. This domain, which is believed to be exposed on the nucleosome surface, is also the most evolutionarily divergent region of the L. infantum histone H3. Visceral leishmaniasis (VL) sera do not react with mammalian histones, an indication that the anti-histone response elicited during Leishmania infection is triggered by the parasite histone. The results of the prevalence of anti-histone H3 antibodies in canine VL sera together with the sequence-specific characteristics of the amino-terminal region of L. infantum histone H3 indicate that the recombinant protein rLiH3-Nt may be of use for diagnosis of canine VL.

Amino Acid Sequence↗

Identification of Plasmodium falciparum MSP-1 peptides able to bind to human red blood cells.

To determine amino acid sequences of the Plasmodium falciparum MSP-1 protein that interact with red blood cell membranes in a specific receptor-ligand interaction, 78 sequential peptides, 20 amino acids long and spanning the entire length of the molecule, were synthesized and analysed with a specific binding assay developed for this purpose. Results show that peptides based on conserved and dimorphic regions of MSP-1, interact with human red blood cells (RBCs). This interaction occurs predominantly with peptides contained within the MSP-1 proteolytic fragments of 83 kDa, 38 kDa, 33 kDa and 19 kDa. Affinity constants of these peptides were between 140 and 250 nM. Peptide-RBC binding post enzyme treatment showed that the RBC receptors are not sialic acid dependent and appear to be proteic in nature. Some of these peptides inhibited merozoite invasion of RBCs yet did not inhibit intraerthrocytic development. These peptides, in conjunction with those from other merozoite surface proteins, may be used to rationally design a second generation of synthetic peptide-based malaria vaccines.

Amino Acid Sequence↗

Diagnosis of cutaneous tuberculosis by polymerase chain reaction using a species-specific gene.

BACKGROUND: Diagnosis of tuberculosis (TB), especially cutaneous TBC, by conventional microbiologic methods is still a very laborious process and the results are usually inconclusive. Our purpose was to identify M. tuberculosis bacilli in uncultured clinical samples from skin lesions by means of the rapid, specific, and sensitive polymerase chain reaction (PCR). METHODS: The PCR, using a set of species-specific primers, was performed on biopsies and fluid secretions from lesions. RESULTS: A positive amplification reaction was observed in three of the four samples studied. For one of the samples, the result was confirmed by a positive culture in Löwenstein-Jensen medium and for the other two, by molecular hybridization and the clinical course of the patients after treatment. Samples obtained from a patient with panniculitis of Christian-Weber and a normal skin biopsy were included as negative controls. CONCLUSIONS: We propose the PCR method as a tool for the diagnosis of cutaneous TBC. The presence of the M. tuberculosis in an erythema induratum of Bazin suggests a revision of the concept of this disease as a tuberculide reaction.

Adult↗

Multiprimer PCR system for differential identification of mycobacteria in clinical samples.

A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obtained, using reference mycobacterial strains and typed clinical isolates, show that the multiprimer PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay.

Base Sequence↗

Human heart-infiltrating T-cell clones from rheumatic heart disease patients recognize both streptococcal and cardiac proteins.

BACKGROUND: beta-Hemolytic streptococcal infection in developing countries still causes thousands of causes of rheumatic heart disease, demanding surgical valve correction. Antigenic mimicry between self and streptococcal components has been proposed as the triggering factor leading to autoimmunity in individuals with genetic susceptibility. Although heart streptococcal-M protein cross-reactive antibodies have been demonstrated, heart tissue damage seems to be T lymphocyte-dependent. We studied the infiltrating T lymphocytes in rheumatic heart lesions with the aim of understanding the role of cellular immune response at the site of the lesions. METHODS AND RESULTS: We obtained 107 T-cell clones from surgical fragments of cardiac tissue from four rheumatic heart disease patients. We tested their capacity to recognize streptococcal M protein-derived synthetic peptides and heart proteins. We found eight infiltrating T-cell clones from all four patients that simultaneously recognize streptococcal M and heart proteins. Among the M-protein sequences tested, only synthetic peptides corresponding to regions 1 through 25, 81 through 103, and 163 through 177 were simultaneously recognized with heart protein fractions. Interestingly, regions 81 through 103 and 163 through 177 have been known to bear heart cross-reactive epitopes at the antibody level. Five of these clones are CD4+, and one is CD8+. CONCLUSIONS: The presence of heart-M protein cross-reactive T-cell clones in rheumatic heart lesions suggests their direct involvement in the pathogenesis of this disease. The dissection of protective and pathogenic epitopes of streptococcal M protein is an important step in allowing the development of a safe anti-streptococcal synthetic vaccine.

Amino Acid Sequence↗

Autoimmunity in Chagas disease cardiopathy: biological relevance of a cardiac myosin-specific epitope crossreactive to an immunodominant Trypanosoma cruzi antigen.

Heart tissue destruction in chronic Chagas disease cardiopathy (CCC) may be caused by autoimmune recognition of heart tissue by a mononuclear cell infiltrate decades after Trypanosoma cruzi infection. Indirect evidence suggests that there is antigenic crossreactivity between T. cruzi and heart tissue. As there is evidence for immune recognition of cardiac myosin in CCC, we searched for a putative myosin-crossreactive T. cruzi antigen. T. cruzi lysate immunoblots were probed with anti-cardiac myosin heavy chain IgG antibodies (AMA) affinity-purified from CCC or asymptomatic Chagas disease patient-seropositive sera. A 140/116-kDa doublet was predominantly recognized by AMA from CCC sera. Further, recombinant T. cruzi protein B13--whose native protein is also a 140- and 116-kDa double band--was identified by crossreactive AMA. Among 28 sera tested in a dot-blot assay, AMA from 100% of CCC sera but only 14% of the asymptomatic Chagas disease sera recognized B13 protein (P = 2.3 x 10(-6)). Sequence homology to B13 protein was found at positions 8-13 and 1442-1447 of human cardiac myosin heavy chain. Competitive ELISA assays that used the correspondent myosin synthetic peptides to inhibit serum antibody binding to B13 protein identified the heart-specific AAALDK (1442-1447) sequence of human cardiac myosin heavy chain and the homologous AAAGDK B13 sequence as the respective crossreactive epitopes. The recognition of a heart-specific T. cruzi crossreactive epitope, in strong association with the presence of chronic heart lesions, suggests the involvement of crossreactivity between cardiac myosin and B13 in the pathogenesis of CCC.

Amino Acid Sequence↗

Identification of the Leishmania infantum P0 ribosomal protein epitope in canine visceral leishmaniasis.

In the present work we show that a high percentage of the sera from dogs naturally affected with viscero-cutaneous leishmaniasis contain antibodies reacting with the Leishmania infantum P0 ribosomal protein. In order to map the antigenic determinants of the LiP0 protein during Leishmania-infection, the complete amino acid sequence of the protein was synthesized as overlapping 20-mer peptides. We have identified the sequence AAKEEPEESDEDDFGMG, located adjacent to the C-terminal end of the protein, as the major antigenic determinant. The anti-LiP0 antibodies present in the sera of the infected dogs do not cross-react with a relatively similar antigenic determinant of the LiP2 acidic proteins as an indication that the Leishmania P0 protein is an independent immunogenically functional antigen in the canine form of the infection.

Amino Acid Sequence↗

Mapping of the linear antigenic determinants from the Leishmania infantum histone H2A recognized by sera from dogs with leishmaniasis.

Antibodies reacting against the H2A histone protein were frequently observed in the sera from dogs naturally infected with the protozoan parasite Leishmania infantum. Using synthetic peptides covering the complete sequence of the protein we have identified the amino terminal region, comprising from amino acids 1 to 20, and the carboxyl terminal region, comprising from amino acids 106 to 132, as conforming the antigenic determinants of the protein. Those regions, exposed in the nucleosome surface, are highly divergent in sequence relative to the mammalian H2A histones. The anti-H2A histone antibodies present in the sera of these dogs specifically recognize the L. infantum H2A histone and they do not react with mammalian histones. The present data indicate that, in spite of the evolutionary conservation of the H2A histone protein among eukaryotic organisms, the humoral response against this protein during natural infection is specifically triggered by the parasite protein antigenic determinants.

Amino Acid Sequence↗

Malaria vaccines.

Explore the source record for details and available documents.

Forecasting↗

Species-specific identification of Mycobacterium bovis by PCR.

The Random Amplified Polymorphic DNA (RAPD) technique was used in the identification of a species-specific fragment of Mycobacterium bovis. A fragment of approximately 500 bp was amplified from the genome of 15 different M. bovis strains, including M. bovis BCG Pasteur, but was shown to be absent in 26 different mycobacteria and 20 different clinical isolates of Mycobacterium tuberculosis. When the fragment was used as a probe in a Southern blot analysis, several radioactive bands common to M. tuberculosis and M. bovis were observed. However, this fragment hybridized specifically to a 2900 bp EcoRI fragment in the M. bovis genome, but failed to hybridize in either M. tuberculosis or M. avium chromosomal DNA. Based on a partial nucleotide sequence of the 500 bp fragment, two oligonucleotide primers were designed and a PCR assay was developed. Using purified mycobacterial DNA samples, only M. bovis and M. bovis BCG rendered a unique amplification band. This PCR assay is able to detect down to 10 fg purified M. bovis DNA, which corresponds roughly to two bacilli. The assay is also useful for identifying the bacilli directly from uncultured biological samples, such as milk.

Bacterial Typing Techniques↗

During active viscerocutaneous leishmaniasis the anti-P2 humoral response is specifically triggered by the parasite P proteins.

In this work we show that in the sera from dogs naturally infected with the protozoan parasite Leishmania infantum there are antibodies that react specifically against the parasite acidic ribosomal proteins LiP2a and LiP2b, and that each one of the Leishmania P proteins elicits a specific humoral immune response. Using synthetic peptides, the antigenic epitope of these proteins has been mapped in a single region located adjacent to the C-terminal domain highly conserved among the eukaryotic P proteins. The anti-P antibodies elicited during the Leishmania infection do not recognize the conserved C-terminal domain of the parasite P proteins, in contrast with the findings reported in Chagas' disease or systemic lupus erythematosus. The antigenic epitopes of the LiP2a and LiP2b are almost identical in amino acid sequence. No reactivity against Trypanosoma cruzi and human P proteins was found in sera from L. infantum-infected dogs.

Amino Acid Sequence↗

A species-specific nucleotide sequence of Mycobacterium tuberculosis encodes a protein that exhibits hemolytic activity when expressed in Escherichia coli.

Species-specific proteins may be implicated in the unique pathogenic mechanisms characteristic of Mycobacterium tuberculosis. In previous studies, a 3.0-kb species-specific DNA fragment of M. tuberculosis was identified (C. A. Parra, L. P. Londoño, P. del Portillo, and M. E. Patarroyo, Immun. 59:3411-3417, 1991). The nucleotide sequence of this 3.0-kb fragment has been obtained. This sequence was shown to contain two open reading frames (ORFs) whose putative gene products share 68.9% identity between each other. The major ORF shows 57.8% similarity with PLC-N and 53.2% similarity with PLC-H, two phospholipase C enzymes from Pseudomonas aeruginosa. The major ORF was amplified by PCR and cloned into the pGEX-5T expression vector. Cell extracts of Escherichia coli overexpressing this glutathione S-transferase fusion protein were shown to produce beta-hemolysis suggestive of phospholipase activity. Since phospholipase C enzymes have been reported as virulence factors of P. aeruginosa and also of the intracellular pathogen Listeria monocytogenes, it is possible that the proteins identified in this study could also play a role in sustaining tuberculosis infection in humans.

Amino Acid Sequence↗

Mapping of antigenic determinants of the T. cruzi hsp70 in chagasic and healthy individuals.

In the present paper we describe the analysis of the immunological recognition by sera of healthy individuals and chagasic patients of the Trypanosoma cruzi heat shock 70 kDa protein. By a Falcon Assay Screening Test, using as antigen an ATP-agarose purified T. cruzi hsp70, it has been found that the sera of infected patients as well as of that of healthy individuals show reactivity against the hsp70 protein but that the reactivity of the sera of patients is in general significantly higher than that of healthy individuals. The analysis of the reactivity of the chagasic sera against a collection of peptides covering 92% of the protein has shown that more than 50% of the peptides gave a positive response but only against a few peptides did we observe high reactivity in a wide spectrum of sera. Only four peptides (numbers 9, 12, 14 and 47) were recognized by all sera tested with high reactivity values. The sera of healthy individuals also showed reactivity against a large percentage of peptides but with lower values. It was observed that particular peptides showing high reactivity against the sera of healthy donors also show high reactivity against patients' sera. However, the general pattern of reactivity against the peptides is different in chagasic and healthy sera. The immunodominant peptides map in the highly conserved as well as in the less conserved part of the hsp70 molecule. The 1/3 C-terminal, being the least conserved part of the molecule, seems to be the least immunogenic. Mapping of the epitopes led to the identification of particular immunogenic motifs within individual peptides.

Amino Acid Sequence↗

Immunological and functional characterization of proteins of the Mycobacterium tuberculosis antigen 85 complex using synthetic peptides.

As tuberculosis re-emerges as an important health problem worldwide, new drugs, better diagnostic tests and vaccines are being sought. In order to identify potentially useful peptides for the development of a synthetic vaccine against tuberculosis, immunological and functional studies were performed using proteins of the antigen 85 complex. Western blot (immuno-blot) analysis and a lymphoproliferation study was used to investigate the B- and T-cell immune response of tuberculosis patients, healthy household contacts and normal controls to proteins of the Mycobacterium tuberculosis antigen 85 complex. Peptides derived from the 85A amino acid sequence were synthesized and used in fibronectin-binding and in ELISA assays. A peptide with the sequence CQPACRKAGCQTYKWEC bound to radiolabelled fibronectin in a time-dependent manner and was recognized by human sera in ELISA. This peptide was identified as a potential component of a synthetic vaccine against tuberculosis.

Amino Acid Sequence↗