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J M Requena

Publications and source records attributed to J M Requena.

At least 19 recordsLinked to original sources
PubMed2006

Genomic organization and expression of the HSP70 locus in New and Old World Leishmania species.

Heat shock is believed to be a developmental inductor of differentiation in Leishmania. Furthermore, heat shock genes are extensively studied as gene models to decipher mechanisms of gene regulation in kinetoplastids. Here, we describe the organization and expression of the HSP70 loci in representative Leishmania species (L. infantum, L. major, L. tropica, L. mexicana, L. amazonensis and L. braziliensis). With the exception of L. braziliensis, the organization of the HSP70 loci was found to be well conserved among the other Leishmania species. Two types of genes, HSP70-I and HSP70-II, were found to be present in these Leishmania species except for L. braziliensis that lacks HSP70-II gene. Polymorphisms in the HSP70 locus allow the differentiation of the Old and New World species within the subgenus Leishmania. A notable discrepancy between our data and those of the L. major genome database in relation to the gene copy number composing the L. major HSP70 locus was revealed. The temperature-dependent accumulation of the HSP70-I mRNAs is also conserved among the different Leishmania species with the exception of L. braziliensis. In spite of these differences, analysis of the HSP70 synthesis indicated that the HSP70 mRNAs are also preferentially translated during heat shock in L. braziliensis.

3' Untranslated Regions↗
PubMed2006

Immunohistological features of visceral leishmaniasis in BALB/c mice.

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.

Animals↗
PubMed2003

A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L. infantum.

The capacity of a quimeric protein, formed by the genetic fusion of five antigenic determinants from four Leishmania proteins, formulated with BCG, to protect dogs against Leishmania infantum infection is described. The data showed that after i.v. administration of 500,000 parasites of the L. infantum M/CAN/ES/96/BCN150 strain, zymodeme MON-1, the animals became infected as suggested by the humoral response against the parasite antigens. All control unvaccinated dogs had parasites in the lymph nodes at day 150 post-infection. One of these unvaccinated infected dog was parasite negative at day 634 behaving, thus, as resistant. In contrast, only 50% of the immunized dogs had parasites in the lymph nodes at day 150 post-infection. Four of these dogs became parasite negative by day 634 post-infection. The control animals developed at various times during the follow-up period clinical symptoms associated with Leishmaniasis. The control diseased dogs developed also in the liver and spleen some of the abnormal histological features associated with natural visceral Leishmaniasis. The immunized dogs, however, were not only normal at the clinical but also at the anatomo-pathological level. A positive delayed type hypersensitivity (DTH) response was observed in nine of the immunized protected dogs. The data indicated that Q+BCG confers 90% protection against infection and at least 90% protection at the clinical level.

Animals↗
PubMed2003

Leishmania infantum possesses a complex family of histone H2A genes: structural characterization and analysis of expression.

We have studied the genomic organization and transcription of the histone H2A genes in the protozoan parasite Leishmania infantum. In the parasite genome 2 gene clusters exist, each containing 3 H2A gene copies. Sequence analyses showed the existence of significant sequence divergence among the H2A genes, mainly in their 5'- and 3'-untranslated regions (UTRs). Also, the existence of allelic alternatives has been evidenced. Based on the divergence in the 3'UTR regions, we have defined 3 classes of H2A transcripts, which are present at different levels in L. infantum promastigotes. However, transcription of the 3 classes of H2A genes occurs at similar levels, as measured by nuclear run-on assays, indicating that their abundance is regulated post-transcriptionally. Also, differences in regulation were observed among the H2A transcripts: the levels of transcripts with 3'-UTR type I and type III are affected by growth phase whereas transcripts with 3'-UTR type II, that are barely detected, remain constant. It is likely that the complexity, in both gene organization and differential expression exhibited by the L. infantum H2A genes, is imposed by the nature of the post-transcriptional mechanisms of regulation operating in this parasite.

Amino Acid Sequence↗
PubMed2001

Analysis of the adjuvant effect of recombinant Leishmania infantum Hsp83 protein as a tool for vaccination.

The properties of Leishmania infantum hsp83 (LiHsp83) to elicit an immune response against a fused reporter antigen, maltose binding protein (MBP), was studied. CF1 mice were immunized with different purified recombinant proteins: MBP, LiHsp83 and MBP fused to LiHsp83 (MBP-LiHsp83). Serum samples were obtained at days 0, 21, 28, 60, 90, 120 and 150 post-immunization. MBP-LiHsp83 fusion protein elicited a strong humoral response against MBP, higher than that one obtained in mice immunized with MBP alone or MBP mixed with LiHsp83, showing the secretion of both anti-MBP IgG2a and IgG1 isotypes (IgG2a/IgG1 ratio: 2:1). This response was specific for recombinant proteins and was maintained for at least 150 days, whereas the reactivity in mice immunized with MBP alone dissapeared at day 90. After in vitro stimulation with MBP, spleen cells from MBP-LiHsp83 immunized mice showed higher proliferation indices and produced higher secretion of IFN-gamma than spleen cells from either control or MBP-immunized mice. In all groups of mice IL-4 was undetectable. Thus we consider that LiHsp83 may be a promising candidate to be used as carrier of fused antigens for adjuvant-free vaccination.

Adjuvants, Immunologic↗
PubMed2001

Characterisation of a monoclonal antibody recognising specifically the HSP70 from Leishmania.

Heat-shock protein 70 (HSP70) is ubiquitously distributed along the evolutionary scale and has such an amino acid sequence conservation that it is considered the most evolutionarily conserved protein. In order to obtain immunological tools specific against Leishmania infantum HSP70, hybridomas were established that secreted monoclonal antibodies (mAbs) against recombinant L. infantum HSP70. One of them, named mAb 2B8D2, specifically reacted with the Leishmania protein and did not recognise HSP70 from the related kinetoplastid Trypanosoma cruzi. The use of synthetic peptides allowed us to determine the B-cell epitope recognised by this mAb, an epitope located in the divergent C-terminal domain of the protein. Remarkably, the mAb possesses the capacity to immunoprecipate HSP70 from promastigote extracts of L. infantum. The fact that human HSP70 is not recognised by this mAb assures the usefulness of this antibody for diagnostic purposes and studies involving Leishmania infection of macrophages.

Amino Acid Sequence↗
PubMed2000

Immune and clinical parameters associated with Leishmania infantum infection in the golden hamster model.

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.

Animals↗
PubMed2000

Histone synthesis in Leishmania infantum is tightly linked to DNA replication by a translational control.

We have analysed the regulation of histone synthesis in Leishmania infantum following inhibition of DNA replication. Run-on experiments indicated that transcription rates of the genes coding for the four core histones (H2A, H2B, H3 and H4) were not affected by the inhibition with hydroxyurea of DNA synthesis. However, a dramatic decrease was observed in the newly synthesized histones after inhibition of DNA synthesis. Furthermore, the synthesis of both the histones and DNA resumed in promastigotes after removal of hydroxyurea, indicating that inhibition was reversible. Unlike most eukaryotes, in which the replication-dependent histone transcripts decrease upon a replication blockade, the levels of L. infantum histone mRNAs do not change under similar conditions. Thus the present data indicate that histone synthesis in Leishmania is tightly coupled to DNA replication by a mechanism operating at the translational level.

Animals↗
PubMed2000

Genomic clustering of the Trypanosoma cruzi nonlong terminal L1Tc retrotransposon with defined interspersed repeated DNA elements.

We have analyzed the genomic distribution and organization of the long interspersed nucleotide element (LINE) L1Tc, a nonlong terminal repeat (LTR) retrotransposon of Trypanosoma cruzi. The results indicate that the L1Tc element is dispersed along the parasite genome and that in some regions it is organized in tandem repeats. The data allowed us to define the existence of short direct-repeated sequences flanking the genomic L1Tc elements. Relevant is the finding that the LINE L1Tc is located in genomic regions rich in short interspersed nucleotide elements (SINE)-like sequences. In particular, the L1Tc element is found associated to E13-related sequences, redefined in this work and renamed RS13Tc, and to a newly described RS1Tc sequence. The RS1Tc sequence is present, per haploid genome, in about 3,200 copies. Northern blot analysis showed that the RS1Tc is being transcribed into RNAs of different sizes. The analysis of the chromosomal distribution of these elements in various strains of T. cruzi suggested that this type of clustering might be a common feature of the genome of these parasites.

Animals↗
PubMed2000

Leishmania infantum: gene cloning of the GRP94 homologue, its expression as recombinant protein, and analysis of antigenicity.

The complete nucleotide sequence for the Leishmania infantum homologue to the glucose-regulated protein 94 (GRP94) gene was determined from the isolation and characterization of a genomic clone. Like the mammalian and plant GRP94s, the L. infantum GRP94 sequence possesses both an N-terminal signal peptide and a putative endoplasmic reticulum retention signal, consisting of the C-terminal tetrapeptide EDDL. Thus, L. infantum is the first protozoan organism in which GRP94 has been identified. Southern blot analysis has indicated that this protein is encoded by a single-copy gene. The L. infantum GRP94 gene was expressed in Escherichia coli and the recombinant protein used to evaluate its antigenicity and immunogenicity. Eighty-four percent of sera from dogs with visceral leishmaniasis reacted with the protein, indicating that GRP94 is a potent immunogen during Leishmania infection. Given the immunogenic and antigenic properties shown by the L. infantum GRP94, we think that this protein constitutes a valuable molecule for diagnostic purposes and a potential candidate for studies of protective immunogenicity.

Amino Acid Sequence↗
PubMed2000

Identification of a putative regulatory element in the 3'-untranslated region that controls expression of HSP70 in Leishmania infantum.

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.

3' Untranslated Regions↗
PubMed2000

Evolutionarily conserved proteins as prominent immunogens during Leishmania infections.

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.

Animals↗
PubMed2000

The Leishmania infantum acidic ribosomal protein LiP2a induces a prominent humoral response in vivo and stimulates cell proliferation in vitro and interferon-gamma (IFN-gamma) production by murine splenocytes.

The acidic ribosomal proteins of the protozoan parasite Leishmania infantum have been described as prominent antigens during both human and canine visceral leishmaniasis. In this study we present data showing that the intraperitoneal administration in BALB/c mice of the Leishmania LiP2a protein, in the absence of any added adjuvants, elicited a strong humoral response as an indication that the protein is a potent immunogen. Despite the evolutionary conservation of the acidic ribosomal proteins, the antibody response was found to be specific for the Leishmania protein. Another remarkable finding was the observation that the LiP2a protein stimulates the in vitro proliferation of splenocytes from either LiP2a-immunized or naive BALB/c mice. Since similar proliferative indices were observed in T cell-enriched cultures, it is likely that the LiP2a stimulating activity is due mainly to T lymphocyte expansion. Also, the stimulatory effect was demonstrated to be antigen-specific, since the proliferation was abrogated by the presence of anti-LiP2a antibodies. Interestingly, the LiP2a protein stimulated the production of substantial amounts of IFN-gamma in cultured splenocytes from LiP2a-immunized mice. Our data indicate therefore that the immunostimulatory properties shown by this antigen should be taken into account when developing therapeutic and prophylactic vaccines against leishmaniasis.

Amino Acid Sequence↗
PubMed1999

Phage recovery by electroporation of naked DNA into host cells avoids the use of packaging extracts.

In this paper, we describe the application of electroporation to deliver phage DNA into bacterial cells in order to recover it as phage particles. The methodology represents a quicker and cheaper alternative to the use of packaging extracts to rescue phage clones stored as naked DNAs. Furthermore, our data demonstrate that there were not rearrangements or recombinations between phage DNAs when a mixture of different DNAs was electroporated, suggesting the use of electroporation as a reliable method for construction of gene libraries.

Bacteriophage lambda↗
PubMed1999

Analysis of the humoral immune response against total and recombinant antigens of Leishmania infantum: correlation with disease progression in canine experimental leishmaniasis.

Leishmaniasis by Leishmania infantum in the Mediterranean Basin constitutes an important problem in both human and veterinary medicine. Based in both the importance of canids as reservoirs for the human disease and the fact that the canine disease may be an excellent model for the human condition, the present work has been conducted to analyze clinical and immune mechanisms associated with canine experimental leishmaniasis. Six-month-old mixed-breed dogs were intravenously infected with L. infantum promastigotes and the infection course was monitored along a 343 days-period. On day 75 post-infection (p.i.), amastigotes were observed in the lymph nodes of all dogs. The analysis of the humoral response against total L. infantum antigens by both ELISA and Western blotting evidenced a correlation between the levels of IgG isotypes (IgG1 and IgG2) and disease progression. It was observed that in those animals showing either a regressive or an oligosymptomatic form of the disease, the anti-Leishmania IgG1 antibodies were undetectable whereas those animals developing active disease showed high levels of anti-Leishmania IgG1 antibodies. Additionally, the time-course of antibody production against L. infantum recombinant antigens in the experimentally infected dogs has been analyzed. The present data suggest that reactivity against the heat-shock protein 70 (HSP70) may be used as diagnostic marker of early steps of infection, and that the appearance of anti-histone antibodies is associated with progression of infection to disease status.

Animals↗
PubMed1999

Immunostimulatory properties of the Leishmania infantum heat shock proteins HSP70 and HSP83.

Emerging evidence indicates that the heat shock proteins (HSPs), a set of highly evolutionary conserved proteins, are playing essential roles in both normal processes of the immune system and specific immune responses. In a previous work, we demonstrated that the Leishmania infantum HSP70 possesses remarkable immunostimulatory properties. In the present work, we have extended the study to another HSP from this parasite, the HSP83. We show that this protein also has an adjuvant effect to an accompanying protein by stimulation of the humoral response when both proteins are fused and co-administered to BALBjc mice. The analysis of the IgG isotypes, IgG1 and IgG2a, indicated that the immunisations with the Leishmania HSPs, mainly the HSP70, potentiate a Thl-type response. It was found that the amino-terminal domain of the HSP70, the most evolutionary conserved region of the molecule, maintains the ability to stimulate the humoral response, whereas the carboxyl-terminal domain does not have a similar effect. Unexpectedly, we found that the L. infantum HSP70 and HSP83 recombinant proteins stimulated the proliferation of spleen cells from unprimed BALB/c mice. Remarkably, this proliferation was abolished either by thermal denaturing of the proteins or by using specific antibodies. The use of the T-cell inhibitor cyclosporin A in the splenocytes proliferation assays suggested that both T- and non-T-cells are stimulated by the Leishmania HSPs. These findings may be relevant for therapeutic and prophylactic applications.

Adjuvants, Immunologic↗
PubMed1999

Antigenicity of the Leishmania infantum histones H2B and H4 during canine viscerocutaneous leishmaniasis.

In this study we show that sera from dogs naturally infected with Leishmania infantum contain antibodies that specifically react against the parasite H2B and H4 histones. The Leishmania H2B and the amino-terminal region of the histone H4, expressed as fusion proteins, when confronted with sera from canine viscerocutaneous leishmaniasis (VCL) dogs, were recognized by 63% and 47%, respectively. No reactivity was detected when sera from dogs naturally infected with pathogens other than Leishmania were used. Using a collection of synthetic peptides covering the complete sequence of both proteins, we have determined that the main linear antigenic determinants are located in the amino-terminal domains of these histones. The humoral response against histones H2B and H4 induced during canine leishmaniasis was found to be specific for Leishmania histones, since no cross-reactivity of the VCL sera with mammal histones was observed. Also, a comparative study of the prevalence of antibodies among VCL sera against the four core histones of L. infantum was performed. Although a large heterogeneity of the humoral responses against these proteins was found, histones H2A and H3 seem to be more prevalent immunogens than histones H2B and H4 during canine natural leishmaniasis. The origin of the anti-histone humoral response and its possible implications in the pathogenesis of Leishmania infection are discussed.

Amino Acid Sequence↗