Manuel Soto - Academia.edu (original) (raw)

Papers by Manuel Soto

Research paper thumbnail of Efficient 5′-End Labeling of Oligonucleotides Containing Self-Complementary Sequences

Research paper thumbnail of Organization, transcription and regulation of the Leishmania infantum histone H3 genes

Biochemical Journal, 1996

The genomic organization and transcription of the genes encoding the histone H3 of the protozoan ... more The genomic organization and transcription of the genes encoding the histone H3 of the protozoan parasite Leishmania infantum have been studied. It was found that there are multiple copies of the histone H3 genes distributed in chromosomal bands XIX and XIV. The nucleotide sequence of two of the L. infantum H3 genes, each one located in a different chromosome, is reported. Although the nucleotide sequence of the coding region of both genes is identical, the sequence of the 3´ untranslated region is highly divergent. It was found also that there exist two different size classes of histone H3 transcripts, each one derived from a different gene, and that they are polyadenylated. The steady-state level of the transcripts dramatically decreases when the parasites enter the stationary phase of growth, suggesting a mode of regulation which is linked to the proliferation status of the cell. Unlike the replication-dependent histones, the L. infantum H3 mRNA levels do not decrease after treat...

Research paper thumbnail of Genes and Chromosomes of Leishmania infantum

Memórias do Instituto Oswaldo Cruz, 1997

During recent years, several Leishmania infantum genes have been cloned and characterized. Here, ... more During recent years, several Leishmania infantum genes have been cloned and characterized. Here, we have summarized the available information on the gene organization and expression in this protozoan parasite. From a comparative analysis, the following outstanding features were found to be common to most of the genes characterized: tandemly organized genes with conserved coding regions and divergent untranslated regions, polycistronic transcription and post-transcriptional regulation of gene expression. The analysis of chromosomes of L. infantum by pulsed-field electrophoresis showed the existence of both size and number polymorphisms such that each strain has a distinctive molecular karyotype. Despite this variability, highly conserved physical linkage groups exists among different strains of L. infantum and even among Old World Leishmania species. Gene mapping on the L. infantum molecular karyotype evidenced a bias in chromosomal distribution of, at least, the evolutionary conserved genes.

Research paper thumbnail of Immune and clinical parameters associated with Leishmania infantum infection in the golden hamster model

Veterinary Immunology and Immunopathology, 2000

For experimental infections with viscerotropic strains of Leishmania, a suitable animal model is ... more For experimental infections with viscerotropic strains of Leishmania, a suitable animal model is not yet defined. In the present work, we have reappraised the use of golden hamster (Mesocricetus auratus) as an experimental model for infection with Leishmania infantum. Groups of hamsters were challenged by the intracardial route with doses ranging from 10(3) to 10(5) infectious promastigotes and the animals were monitored for 1-year follow-up period. The outcome of the infection was assessed by clinical symptoms of leishmaniasis, parasite loads in both liver and spleen, humoral response to Leishmania antigens and antibody levels in kidneys. The humoral response was analysed using either crude antigens (by ELISA and Western blotting) or several recombinant Leishmania antigens (Hsp70, Hsp83, LiP2a, LiP2b, H2A, H3 and KMP-11). From the analysis of all these parameters, we established the existence of three groups of animals: symptomatic or susceptible, oligosymptomatic, and resistant. Given the parallelism existing between the outcomes of Leishmania-infection in hamsters, dogs and humans, we believe that our data illustrate that the hamster is an excellent experimental model to study visceral leishmaniasis and for the design of vaccine development.

Research paper thumbnail of Vaccination with a plasmid DNA cocktail encoding the nucleosomal histones of Leishmania confers protection against murine cutaneous leishmaniosis

Vaccine, 2004

Leishmania histones are relevant immunogens for the host immune system during both Leishmania inf... more Leishmania histones are relevant immunogens for the host immune system during both Leishmania infection and disease. In the present paper we have evaluated the prophylactic value of the four Leishmania infantum histones forming the nucleosomal core in the murine model of cutaneous leishmaniasis. In a first stage, the immune response elicited by the intramuscular injection of a mixture of four plasmid DNAs, encoding the L. infantum histones H2A, H2B, H3 and H4, was determined in BALB/c mice. It was found that the immunized animals developed a specific Th1 immune response, which was associated with an antigen-specific production of interferon (IFN-␥) and a limited humoral response against histones (dominated by antibodies of the IgG2a isotype). According to the pure Th1-type immune response elicited by the DNA vaccination with Leishmania histones, vaccinated mice showed a solid immunity that efficiently controlled the Leishmania major infection. The protection in mice vaccinated with histone-DNAs was associated with a low humoral response against leishmanial antigens, an enhanced IFN-␥ production and little, if any, IL-4 production. The relative contribution of both CD8 + and CD4 + T cells to the IFN-␥ production, and the IL-12 dependence were also evaluated. All these data indicated that DNA vaccination with Leishmania histones genes results in a specific Th1-like response during L. major infection, and that both CD4 + and CD8 + T cells contribute to the resistance of vaccinated mice to cutaneous leishmaniasis.

Research paper thumbnail of Evolutionarily Conserved Proteins as Prominent Immunogens during Leishmania Infections

Parasitology Today, 2000

Many Leishmania antigens have been identified as members of conserved protein families, such as t... more Many Leishmania antigens have been identified as members of conserved protein families, such as the acidic ribosomal proteins, the histones and the heat-shock proteins; despite this, they elicit specific immune responses. Furthermore, homologues of many of these antigens are immune targets in other infectious diseases and systemic autoimmune diseases. Here, Jose Mar a Requena, Carlos Alonso and Manuel Soto review this class of widely distributed antigens, which they call 'panantigens'. They also propose a model to explain the prominent immunogenicity of these antigens during Leishmania infection, on the basis of the fact that many panantigens are constituents of multicomponent complexes in the cell. The elucidation of the pathways by which Leishmania antigens are processed and presented to effector cells from the host immune system will shed light on the immunopathology of leishmaniasis and help in the development of protective immunotherapies.

Research paper thumbnail of Bacteriophage λ: long-term stored stocks ready for lysis

Research paper thumbnail of Immunohistological features of visceral leishmaniasis in BALB/c mice

Parasite Immunology, 2006

It has been reported that the level of protection provided by vaccines against murine visceral le... more It has been reported that the level of protection provided by vaccines against murine visceral leishmaniasis (VL) is low and that progress in research on VL may be due to the lack of appropriate models to study protective immunity. We have analysed the immunohistological features occurring in BALB/c mice after intravenous administration of 10(3), 10(5) and 10(6) parasites of Leishmania infantum. Our results show that in all cases parasite administration leads to the establishment of infection and to the development of quantifiable immunohistological features which are dependent on the inoculum size. This study demonstrates that differences in the parasite challenge result in changes in the evolution of some of the parameters associated with the degree of the infection in the BALB/c model: level of anti-Leishmania antibodies, up-regulation of spleen arginase activity, balance between IFN-gamma and IL-10, extent of lymphoid follicle depletion in the splenic white pulp and ineffective development of hepatic granulomas. Also, and depending on the initial infectious inoculum, the absence of parasites in the bone marrow and the number of mature and empty type granulomas were parameters associated with protection. We think that in this model a challenge of the order of 10(5) parasites should prove useful for vaccine studies against VL.

Research paper thumbnail of Adjuvant guided polarization of the immune humoral response against a protective multicomponent antigenic protein (Q) from Leishmania infantum. A CpG + Q mix protects Balb/c mice from infection

Parasite Immunology, 2004

Research paper thumbnail of The mRNA coding for the nucleosomal protein H2A of Leishmania is polyadenylated and has stem-loops at the 3′ end

Nucleic Acids Research, 1991

Research paper thumbnail of Mapping of antigenic determinants of the T. cruzi HSP70 in chagasic and healthy individuals

Molecular Immunology, 1993

Ah&act-In the present paper we describe the analysis of the immunolo~cal r~ognition by sera of he... more Ah&act-In the present paper we describe the analysis of the immunolo~cal r~ognition by sera of healthy individuals and chagasic patients of the Tryp~osoma 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 l/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.

Research paper thumbnail of Identification of a putative regulatory element in the 3′-untranslated region that controls expression of HSP70 in Leishmania infantum

Molecular and Biochemical Parasitology, 2000

The regulation of HSP70 gene expression in Leishmania infantum, in contrast to most eukaryotes, o... more The regulation of HSP70 gene expression in Leishmania infantum, in contrast to most eukaryotes, occurs by mechanisms that operate exclusively at the post-transcriptional level. During the normal growth of L. infantum promastigotes at 26 degrees C the mRNAs derived from the sixth gene of the HSP70 locus are more abundant than the mRNAs derived from the other five HSP70 genes, but only the latter transcripts accumulate after incubation at 37 degrees C. Here, it was found that the full-length 3'untranslated region (UTR) and downstream sequences of the HSP70 genes are necessary for a correct polyadenylation of both types of transcripts and responsible for the differences in the steady-state levels of the transcripts. Also, it was found that the addition of the 3'-UTR-I (common to the first five genes of the L. infantum HSP70 gene cluster) to a reporter gene is sufficient to achieve an accumulation of the corresponding transcripts at 37 degrees C. This effect was, furthermore, found to be strand dependent. A progressive shortening of the 1063-base 3'-UTR-I has shown that the temperature-dependent accumulation was lost after deletion of 364-nucleotides from the 3' end. In addition, the accumulation of reporter transcripts at 37 degrees C was not observed in a plasmid construct containing an internal deletion (region 699-816) of the 3'-UTR-I. Thus, our data suggest that RNAs derived from L. infantum HSP70 genes 1-5 contain a cis-acting sequence that functions as a positive element during heat shock.

Research paper thumbnail of High-efficiency plating method for Leishmania infantum

Molecular and Biochemical Parasitology, 2003

Research paper thumbnail of Characterization of a highly repeated interspersed DNA sequence of Trypanosoma cruzi: its potential use in diagnosis and strain classification

Molecular and Biochemical Parasitology, 1992

In the present paper we report the characterization of a repetitive DNA sequence from the genome ... more In the present paper we report the characterization of a repetitive DNA sequence from the genome of the Maracay strain of Trypanosoma cruzi. The sequence is 1025 nucleotides long and represent about 7% of the total nuclear DNA of the parasite with a copy number of 1-2 x 10(4) copies per genome. It is also present in a high copy number in several strains of T. cruzi but absent from other trypanosomatids and from the human genome. The DNA sequence is interspersed and present on many chromosomes. We show that the different copies of this repeat present extensive endonuclease restriction polymorphisms. Using 2 conserved oligonucleotide primers derived from the repetitive sequence, together with the polymerase chain reaction technique, the presence of less than 1/30 of a single parasite can be readily detected by ethidium bromide staining.

Research paper thumbnail of Regulation of hsp70 expression in Trypanosoma cruzi by temperature and growth phase

Molecular and Biochemical Parasitology, 1992

The steady-state level of the hsp70 mRNAs of Trypanosoma cruzi cultured at different temperatures... more The steady-state level of the hsp70 mRNAs of Trypanosoma cruzi cultured at different temperatures and growth conditions has been analyzed by Northern blotting. We show that only one size class of hsp70 mRNA, of about 2.2 kb, is transcribed from the hsp70 cluster and that its transcription is constitutive at 28°C. However, after a heat shock treatment at 37°C for 2 h of logarithmically growing parasites, the abundance of the hsp70 mRNA increased about 4-fold. A similar increase was observed at 28~'C when the parasite culture reached the stationary phase of growth. On the other hand, a heat shock at 42°C did not change the steady state level of the 2.2-kb size class of hsp70 mRNA. However, accumulation of transcripts of high molecular weight was detected when stationary growing parasites were cultured at 42°C for 2 h. Also at 37°C the steady state level of the a-and [/-tubulin mRNAs of logarithmically growing parasites exhibited a slight increase but only after a period of 24 h. Analysis by one-dimensional immunoblots of the Hsp70 levels showed that at 37°C the abundance of the protein was 4fold higher than at 28°C. lmmunoblots of high-resolution two-dimensional gel electrophoresis showed, moreover, that various isoforms of this protein are constitutively expressed at 28~C and that some of them have a specific pattern of induction at 37°C. We observed, moreover, that the heat, shock induces the expression of a series of proteins while it causes repression of others.

Research paper thumbnail of Analysis of the antigenic properties of the L. infantum Hsp70: design of synthetic peptides for specific serodiagnosis of human leishmaniasis

Immunology Letters, 1998

The 70-kDa heat shock protein (Hsp70) is a major target of the humoral immune response during Lei... more The 70-kDa heat shock protein (Hsp70) is a major target of the humoral immune response during Leishmania infections. In the present paper, it is shown that 84.6% of sera from mucocutaneous leishmaniasis (MCL) patients and 78.9% of sera from visceral leishmaniasis (VL) patients reacted with the L. infantum Hsp70. The mapping of the antigenic determinants indicated that this protein is highly rich in linear B-epitopes. As the complete protein cannot be used for specific serodiagnosis of VL because it is also recognized by the sera from Chagas' disease patients, a search for specific epitopes recognized by leishmaniasis patients was undertaken. A 50-mer synthetic peptide, located in the most divergent region of the protein, was found to be recognized by a high percentage of leishmaniasis sera and not recognized by chagasic sera. Such a tool would be particularly useful for serodiagnosis of leishmaniasis in geographical areas where mixed infections with Trypanosoma cruzi and Leishmania occurs.

Research paper thumbnail of Characterization of a short interspersed reiterated DNA sequence of Trypanosoma cruzi located at the 3′-end of a poly(A)+ transcript

Gene, 1994

Characterization of a short interspersed reiterated DNA sequence of Trypanosoma cruzi located at ... more Characterization of a short interspersed reiterated DNA sequence of Trypanosoma cruzi located at the 3'-end of a poly(A)+ transcript (Expression analysis; sequence analysis; cDNA isolation; genomic distribution; (TAA)n repeats)

Research paper thumbnail of Recent advances in vaccines for leishmaniasis

Expert Opinion on Biological Therapy, 2004

The observation that recovery from infection with Leishmania confers immunity to reinfection sugg... more The observation that recovery from infection with Leishmania confers immunity to reinfection suggests that control of leishmaniasis by vaccination may be possible. However, there are no vaccines available at present to control any form of leishmaniasis, despite considerable efforts. Studies of the immunopathogenesis and mechanisms of protective immunity, mainly derived from animal models of experimental leishmaniasis, have defined a number of features that should be met by an effective vaccine. In addition, several antigens have been identified that may be potential vaccine candidates, and molecular biological techniques have made them available as recombinant proteins for second-generation vaccines. Furthermore, molecules present in the saliva of Leishmania-transmitting vectors have been demonstrated as valuable candidates for the development of anti-Leishmania vaccines. This review concentrates on the most promising vaccine candidates and highlights new approaches for the development of vaccines. Finally, based on present knowledge, the future prospects for developing an effective vaccine against the different clinical forms of leishmaniasis are discussed.

Research paper thumbnail of Conserved nucleotides surrounding the trans-splicing acceptor site and the translation initiation codon in Leishmania genes

Experimental Parasitology, 2003

Research paper thumbnail of Leishmania infantum: Gene Cloning of the GRP94 Homologue, Its Expression as Recombinant Protein, and Analysis of Antigenicity

Experimental Parasitology, 2000

temperatures, comprise a set of proteins known to play diinfantum: Gene cloning of the GRP94 homo... more temperatures, comprise a set of proteins known to play diinfantum: Gene cloning of the GRP94 homologue, its expression as verse roles, even in unstressed cells, in folding, assembly, recombinant protein, and analysis of antigenicity. Experimental Parasiintracellular localization, secretion, regulation, and degradatology 96, 108-115. The complete nucleotide sequence for the Leishtion of proteins (Feder and Hofmann 1999 and references mania infantum homologue to the glucose-regulated protein 94 (GRP94) gene was determined from the isolation and characterization of a genomic therein). In the past few years, the emergence of compelling clone. Like the mammalian and plant GRP94s, the L. infantum GRP94 evidence has lead to the general acceptance that the HSPs sequence possesses both an N-terminal signal peptide and a putative also play a crucial and critical role in adaptive and innate endoplasmic reticulum retention signal, consisting of the C-terminal immunity (Srivastava et al. 1998). The main HSPs, namely tetrapeptide EDDL. Thus, L. infantum is the first protozoan organism HSP90, HSP70, and HSP60, are among the most evolutionin which GRP94 has been identified. Southern blot analysis has indicated that this protein is encoded by a single-copy gene. The L. infantum arily conserved proteins present in all organisms. Another GRP94 gene was expressed in Escherichia coli and the recombinant remarkable feature of this class of proteins is the existence protein used to evaluate its antigenicity and immunogenicity. Eightyof specialized molecular variants, some of which constitute four percent of sera from dogs with visceral leishmaniasis reacted with the protein families. Among those families the HSP70 family protein, indicating that GRP94 is a potent immunogen during Leishmania infection. Given the immunogenic and antigenic properties shown by

Research paper thumbnail of Efficient 5′-End Labeling of Oligonucleotides Containing Self-Complementary Sequences

Research paper thumbnail of Organization, transcription and regulation of the Leishmania infantum histone H3 genes

Biochemical Journal, 1996

The genomic organization and transcription of the genes encoding the histone H3 of the protozoan ... more The genomic organization and transcription of the genes encoding the histone H3 of the protozoan parasite Leishmania infantum have been studied. It was found that there are multiple copies of the histone H3 genes distributed in chromosomal bands XIX and XIV. The nucleotide sequence of two of the L. infantum H3 genes, each one located in a different chromosome, is reported. Although the nucleotide sequence of the coding region of both genes is identical, the sequence of the 3´ untranslated region is highly divergent. It was found also that there exist two different size classes of histone H3 transcripts, each one derived from a different gene, and that they are polyadenylated. The steady-state level of the transcripts dramatically decreases when the parasites enter the stationary phase of growth, suggesting a mode of regulation which is linked to the proliferation status of the cell. Unlike the replication-dependent histones, the L. infantum H3 mRNA levels do not decrease after treat...

Research paper thumbnail of Genes and Chromosomes of Leishmania infantum

Memórias do Instituto Oswaldo Cruz, 1997

During recent years, several Leishmania infantum genes have been cloned and characterized. Here, ... more During recent years, several Leishmania infantum genes have been cloned and characterized. Here, we have summarized the available information on the gene organization and expression in this protozoan parasite. From a comparative analysis, the following outstanding features were found to be common to most of the genes characterized: tandemly organized genes with conserved coding regions and divergent untranslated regions, polycistronic transcription and post-transcriptional regulation of gene expression. The analysis of chromosomes of L. infantum by pulsed-field electrophoresis showed the existence of both size and number polymorphisms such that each strain has a distinctive molecular karyotype. Despite this variability, highly conserved physical linkage groups exists among different strains of L. infantum and even among Old World Leishmania species. Gene mapping on the L. infantum molecular karyotype evidenced a bias in chromosomal distribution of, at least, the evolutionary conserved genes.

Research paper thumbnail of Immune and clinical parameters associated with Leishmania infantum infection in the golden hamster model

Veterinary Immunology and Immunopathology, 2000

For experimental infections with viscerotropic strains of Leishmania, a suitable animal model is ... more For experimental infections with viscerotropic strains of Leishmania, a suitable animal model is not yet defined. In the present work, we have reappraised the use of golden hamster (Mesocricetus auratus) as an experimental model for infection with Leishmania infantum. Groups of hamsters were challenged by the intracardial route with doses ranging from 10(3) to 10(5) infectious promastigotes and the animals were monitored for 1-year follow-up period. The outcome of the infection was assessed by clinical symptoms of leishmaniasis, parasite loads in both liver and spleen, humoral response to Leishmania antigens and antibody levels in kidneys. The humoral response was analysed using either crude antigens (by ELISA and Western blotting) or several recombinant Leishmania antigens (Hsp70, Hsp83, LiP2a, LiP2b, H2A, H3 and KMP-11). From the analysis of all these parameters, we established the existence of three groups of animals: symptomatic or susceptible, oligosymptomatic, and resistant. Given the parallelism existing between the outcomes of Leishmania-infection in hamsters, dogs and humans, we believe that our data illustrate that the hamster is an excellent experimental model to study visceral leishmaniasis and for the design of vaccine development.

Research paper thumbnail of Vaccination with a plasmid DNA cocktail encoding the nucleosomal histones of Leishmania confers protection against murine cutaneous leishmaniosis

Vaccine, 2004

Leishmania histones are relevant immunogens for the host immune system during both Leishmania inf... more Leishmania histones are relevant immunogens for the host immune system during both Leishmania infection and disease. In the present paper we have evaluated the prophylactic value of the four Leishmania infantum histones forming the nucleosomal core in the murine model of cutaneous leishmaniasis. In a first stage, the immune response elicited by the intramuscular injection of a mixture of four plasmid DNAs, encoding the L. infantum histones H2A, H2B, H3 and H4, was determined in BALB/c mice. It was found that the immunized animals developed a specific Th1 immune response, which was associated with an antigen-specific production of interferon (IFN-␥) and a limited humoral response against histones (dominated by antibodies of the IgG2a isotype). According to the pure Th1-type immune response elicited by the DNA vaccination with Leishmania histones, vaccinated mice showed a solid immunity that efficiently controlled the Leishmania major infection. The protection in mice vaccinated with histone-DNAs was associated with a low humoral response against leishmanial antigens, an enhanced IFN-␥ production and little, if any, IL-4 production. The relative contribution of both CD8 + and CD4 + T cells to the IFN-␥ production, and the IL-12 dependence were also evaluated. All these data indicated that DNA vaccination with Leishmania histones genes results in a specific Th1-like response during L. major infection, and that both CD4 + and CD8 + T cells contribute to the resistance of vaccinated mice to cutaneous leishmaniasis.

Research paper thumbnail of Evolutionarily Conserved Proteins as Prominent Immunogens during Leishmania Infections

Parasitology Today, 2000

Many Leishmania antigens have been identified as members of conserved protein families, such as t... more Many Leishmania antigens have been identified as members of conserved protein families, such as the acidic ribosomal proteins, the histones and the heat-shock proteins; despite this, they elicit specific immune responses. Furthermore, homologues of many of these antigens are immune targets in other infectious diseases and systemic autoimmune diseases. Here, Jose Mar a Requena, Carlos Alonso and Manuel Soto review this class of widely distributed antigens, which they call 'panantigens'. They also propose a model to explain the prominent immunogenicity of these antigens during Leishmania infection, on the basis of the fact that many panantigens are constituents of multicomponent complexes in the cell. The elucidation of the pathways by which Leishmania antigens are processed and presented to effector cells from the host immune system will shed light on the immunopathology of leishmaniasis and help in the development of protective immunotherapies.

Research paper thumbnail of Bacteriophage λ: long-term stored stocks ready for lysis

Research paper thumbnail of Immunohistological features of visceral leishmaniasis in BALB/c mice

Parasite Immunology, 2006

It has been reported that the level of protection provided by vaccines against murine visceral le... more It has been reported that the level of protection provided by vaccines against murine visceral leishmaniasis (VL) is low and that progress in research on VL may be due to the lack of appropriate models to study protective immunity. We have analysed the immunohistological features occurring in BALB/c mice after intravenous administration of 10(3), 10(5) and 10(6) parasites of Leishmania infantum. Our results show that in all cases parasite administration leads to the establishment of infection and to the development of quantifiable immunohistological features which are dependent on the inoculum size. This study demonstrates that differences in the parasite challenge result in changes in the evolution of some of the parameters associated with the degree of the infection in the BALB/c model: level of anti-Leishmania antibodies, up-regulation of spleen arginase activity, balance between IFN-gamma and IL-10, extent of lymphoid follicle depletion in the splenic white pulp and ineffective development of hepatic granulomas. Also, and depending on the initial infectious inoculum, the absence of parasites in the bone marrow and the number of mature and empty type granulomas were parameters associated with protection. We think that in this model a challenge of the order of 10(5) parasites should prove useful for vaccine studies against VL.

Research paper thumbnail of Adjuvant guided polarization of the immune humoral response against a protective multicomponent antigenic protein (Q) from Leishmania infantum. A CpG + Q mix protects Balb/c mice from infection

Parasite Immunology, 2004

Research paper thumbnail of The mRNA coding for the nucleosomal protein H2A of Leishmania is polyadenylated and has stem-loops at the 3′ end

Nucleic Acids Research, 1991

Research paper thumbnail of Mapping of antigenic determinants of the T. cruzi HSP70 in chagasic and healthy individuals

Molecular Immunology, 1993

Ah&act-In the present paper we describe the analysis of the immunolo~cal r~ognition by sera of he... more Ah&act-In the present paper we describe the analysis of the immunolo~cal r~ognition by sera of healthy individuals and chagasic patients of the Tryp~osoma 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 l/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.

Research paper thumbnail of Identification of a putative regulatory element in the 3′-untranslated region that controls expression of HSP70 in Leishmania infantum

Molecular and Biochemical Parasitology, 2000

The regulation of HSP70 gene expression in Leishmania infantum, in contrast to most eukaryotes, o... more The regulation of HSP70 gene expression in Leishmania infantum, in contrast to most eukaryotes, occurs by mechanisms that operate exclusively at the post-transcriptional level. During the normal growth of L. infantum promastigotes at 26 degrees C the mRNAs derived from the sixth gene of the HSP70 locus are more abundant than the mRNAs derived from the other five HSP70 genes, but only the latter transcripts accumulate after incubation at 37 degrees C. Here, it was found that the full-length 3'untranslated region (UTR) and downstream sequences of the HSP70 genes are necessary for a correct polyadenylation of both types of transcripts and responsible for the differences in the steady-state levels of the transcripts. Also, it was found that the addition of the 3'-UTR-I (common to the first five genes of the L. infantum HSP70 gene cluster) to a reporter gene is sufficient to achieve an accumulation of the corresponding transcripts at 37 degrees C. This effect was, furthermore, found to be strand dependent. A progressive shortening of the 1063-base 3'-UTR-I has shown that the temperature-dependent accumulation was lost after deletion of 364-nucleotides from the 3' end. In addition, the accumulation of reporter transcripts at 37 degrees C was not observed in a plasmid construct containing an internal deletion (region 699-816) of the 3'-UTR-I. Thus, our data suggest that RNAs derived from L. infantum HSP70 genes 1-5 contain a cis-acting sequence that functions as a positive element during heat shock.

Research paper thumbnail of High-efficiency plating method for Leishmania infantum

Molecular and Biochemical Parasitology, 2003

Research paper thumbnail of Characterization of a highly repeated interspersed DNA sequence of Trypanosoma cruzi: its potential use in diagnosis and strain classification

Molecular and Biochemical Parasitology, 1992

In the present paper we report the characterization of a repetitive DNA sequence from the genome ... more In the present paper we report the characterization of a repetitive DNA sequence from the genome of the Maracay strain of Trypanosoma cruzi. The sequence is 1025 nucleotides long and represent about 7% of the total nuclear DNA of the parasite with a copy number of 1-2 x 10(4) copies per genome. It is also present in a high copy number in several strains of T. cruzi but absent from other trypanosomatids and from the human genome. The DNA sequence is interspersed and present on many chromosomes. We show that the different copies of this repeat present extensive endonuclease restriction polymorphisms. Using 2 conserved oligonucleotide primers derived from the repetitive sequence, together with the polymerase chain reaction technique, the presence of less than 1/30 of a single parasite can be readily detected by ethidium bromide staining.

Research paper thumbnail of Regulation of hsp70 expression in Trypanosoma cruzi by temperature and growth phase

Molecular and Biochemical Parasitology, 1992

The steady-state level of the hsp70 mRNAs of Trypanosoma cruzi cultured at different temperatures... more The steady-state level of the hsp70 mRNAs of Trypanosoma cruzi cultured at different temperatures and growth conditions has been analyzed by Northern blotting. We show that only one size class of hsp70 mRNA, of about 2.2 kb, is transcribed from the hsp70 cluster and that its transcription is constitutive at 28°C. However, after a heat shock treatment at 37°C for 2 h of logarithmically growing parasites, the abundance of the hsp70 mRNA increased about 4-fold. A similar increase was observed at 28~'C when the parasite culture reached the stationary phase of growth. On the other hand, a heat shock at 42°C did not change the steady state level of the 2.2-kb size class of hsp70 mRNA. However, accumulation of transcripts of high molecular weight was detected when stationary growing parasites were cultured at 42°C for 2 h. Also at 37°C the steady state level of the a-and [/-tubulin mRNAs of logarithmically growing parasites exhibited a slight increase but only after a period of 24 h. Analysis by one-dimensional immunoblots of the Hsp70 levels showed that at 37°C the abundance of the protein was 4fold higher than at 28°C. lmmunoblots of high-resolution two-dimensional gel electrophoresis showed, moreover, that various isoforms of this protein are constitutively expressed at 28~C and that some of them have a specific pattern of induction at 37°C. We observed, moreover, that the heat, shock induces the expression of a series of proteins while it causes repression of others.

Research paper thumbnail of Analysis of the antigenic properties of the L. infantum Hsp70: design of synthetic peptides for specific serodiagnosis of human leishmaniasis

Immunology Letters, 1998

The 70-kDa heat shock protein (Hsp70) is a major target of the humoral immune response during Lei... more The 70-kDa heat shock protein (Hsp70) is a major target of the humoral immune response during Leishmania infections. In the present paper, it is shown that 84.6% of sera from mucocutaneous leishmaniasis (MCL) patients and 78.9% of sera from visceral leishmaniasis (VL) patients reacted with the L. infantum Hsp70. The mapping of the antigenic determinants indicated that this protein is highly rich in linear B-epitopes. As the complete protein cannot be used for specific serodiagnosis of VL because it is also recognized by the sera from Chagas' disease patients, a search for specific epitopes recognized by leishmaniasis patients was undertaken. A 50-mer synthetic peptide, located in the most divergent region of the protein, was found to be recognized by a high percentage of leishmaniasis sera and not recognized by chagasic sera. Such a tool would be particularly useful for serodiagnosis of leishmaniasis in geographical areas where mixed infections with Trypanosoma cruzi and Leishmania occurs.

Research paper thumbnail of Characterization of a short interspersed reiterated DNA sequence of Trypanosoma cruzi located at the 3′-end of a poly(A)+ transcript

Gene, 1994

Characterization of a short interspersed reiterated DNA sequence of Trypanosoma cruzi located at ... more Characterization of a short interspersed reiterated DNA sequence of Trypanosoma cruzi located at the 3'-end of a poly(A)+ transcript (Expression analysis; sequence analysis; cDNA isolation; genomic distribution; (TAA)n repeats)

Research paper thumbnail of Recent advances in vaccines for leishmaniasis

Expert Opinion on Biological Therapy, 2004

The observation that recovery from infection with Leishmania confers immunity to reinfection sugg... more The observation that recovery from infection with Leishmania confers immunity to reinfection suggests that control of leishmaniasis by vaccination may be possible. However, there are no vaccines available at present to control any form of leishmaniasis, despite considerable efforts. Studies of the immunopathogenesis and mechanisms of protective immunity, mainly derived from animal models of experimental leishmaniasis, have defined a number of features that should be met by an effective vaccine. In addition, several antigens have been identified that may be potential vaccine candidates, and molecular biological techniques have made them available as recombinant proteins for second-generation vaccines. Furthermore, molecules present in the saliva of Leishmania-transmitting vectors have been demonstrated as valuable candidates for the development of anti-Leishmania vaccines. This review concentrates on the most promising vaccine candidates and highlights new approaches for the development of vaccines. Finally, based on present knowledge, the future prospects for developing an effective vaccine against the different clinical forms of leishmaniasis are discussed.

Research paper thumbnail of Conserved nucleotides surrounding the trans-splicing acceptor site and the translation initiation codon in Leishmania genes

Experimental Parasitology, 2003

Research paper thumbnail of Leishmania infantum: Gene Cloning of the GRP94 Homologue, Its Expression as Recombinant Protein, and Analysis of Antigenicity

Experimental Parasitology, 2000

temperatures, comprise a set of proteins known to play diinfantum: Gene cloning of the GRP94 homo... more temperatures, comprise a set of proteins known to play diinfantum: Gene cloning of the GRP94 homologue, its expression as verse roles, even in unstressed cells, in folding, assembly, recombinant protein, and analysis of antigenicity. Experimental Parasiintracellular localization, secretion, regulation, and degradatology 96, 108-115. The complete nucleotide sequence for the Leishtion of proteins (Feder and Hofmann 1999 and references mania infantum homologue to the glucose-regulated protein 94 (GRP94) gene was determined from the isolation and characterization of a genomic therein). In the past few years, the emergence of compelling clone. Like the mammalian and plant GRP94s, the L. infantum GRP94 evidence has lead to the general acceptance that the HSPs sequence possesses both an N-terminal signal peptide and a putative also play a crucial and critical role in adaptive and innate endoplasmic reticulum retention signal, consisting of the C-terminal immunity (Srivastava et al. 1998). The main HSPs, namely tetrapeptide EDDL. Thus, L. infantum is the first protozoan organism HSP90, HSP70, and HSP60, are among the most evolutionin which GRP94 has been identified. Southern blot analysis has indicated that this protein is encoded by a single-copy gene. The L. infantum arily conserved proteins present in all organisms. Another GRP94 gene was expressed in Escherichia coli and the recombinant remarkable feature of this class of proteins is the existence protein used to evaluate its antigenicity and immunogenicity. Eightyof specialized molecular variants, some of which constitute four percent of sera from dogs with visceral leishmaniasis reacted with the protein families. Among those families the HSP70 family protein, indicating that GRP94 is a potent immunogen during Leishmania infection. Given the immunogenic and antigenic properties shown by