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C Alonso

Publications and source records attributed to C Alonso.

At least 181 records · Page 10Linked to original sources
PubMed1996

Synthesis, characterization, and DNA modification induced by a novel Pt-berenil compound with cytotoxic activity.

In the present paper, we show that the reaction of the antipyranosomatid berenil drug with K2PtCl4 resulted in the synthesis of a covalent (Pt(II)-berenil compound of formula [Pt2Cl4(berenil)2]Cl4.4H2O as shown by IR, 1H, 13C, and 195Pt-NMR. The Pt-berenil compound was tested for in vitro antitumor activity against HL-60 and U-937 human leukemic cells. The results show that the LC70 values of the Pt-berenil are about two-fold lower than those of cis-DDP in both HL-60 and U-937 cell lines. Melting data of Pt-berenil:DNA and berenil:DNA complexes indicate that the platinated compound produces on a DNA secondary structure higher compaction than the berenil ligand. The mobility in agarose gels and the circular dichroism spectra of the compounds:DNA complexes revealed, moreover, that both induce drastic changes on a DNA secondary and tertiary structure. The total reflection X-ray fluorescence data showed, in additIon that DNA platination in Pt-berenil:DNA complexes occurs within minutes after addition of the drug, in contrast to what that observed in cis-DDP:DNA complexes. On the basis of these results, we propose that in Pt-berenil, the berenil ligand acts as a carrier of the active cis-P(II) centers towards DNA.

Antineoplastic Agents↗
PubMed1996

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↗
PubMed1996

Genomic repetitive DNA elements of Trypanosoma cruzi.

Repetitive DNA sequences are interspersed throughout the genomes of mammals and other higher eukaryotes, and represent a substantial portion of the genome. Although it has been generally assumed that the redundant DNA is present only in the complex genomes of high order organisms, over the past few years a number of repetitive DNA sequences have been also detected in the protozoan parasite Trypanosoma cruzi. A compilation of the repetitive DNA sequences found in the T. cruzi genome is here presented by Jose Maria Requena, Manuel Carlos López and Carlos Alonso, who also speculate on their possible origin and functional implications regarding retrotransposition and gene regulation.

Journal Article↗
PubMed1996

During canine leishmaniasis a protein belonging to the 83-kDa heat-shock protein family elicits a strong humoral response.

By screening of a Leishmania infantum expression library with the serum from a dog affected with visceral leishmaniasis, a cDNA clone with sequence homology to the Hsp83 gene family was isolated. From analysis of the genomic distribution of the cDNA sequence, it was estimated that the L. infantum genome contains 7 Hsp83 genes tandemly organized. The full-length coding region of the Hsp83 gene located at the 5'-end of the cluster was determined. The deduced amino acid sequence of the L. infantum Hsp83 shows a high level of sequence identity with members of the Hsp83's protein family from other eukaryotic organisms. The complete protein (LiHsp83) and 4 subfragments (LiA1, LiB1, LiC1 and LiD1) were expressed in Escherichia coli as recombinant proteins and used as target antigens in FAST-ELISA assays against a collection of sera from dogs with visceral leishmaniasis. Ninety percent of the sera recognized the recombinant LiHsp83, indicating that L. infantum Hsp83 is a potent immunogen during canine leishmaniasis. Serological analysis of the recombinant subfragments identified the LiB1 subfragment, from amino acid 156 to 283, as the immunodominant region of the protein. This region, which is the less evolutionary conserved region of the protein, was recognized by 88% of the visceral leishmaniasis sera. The results suggest that L. infantum Hsp83 and particular protein subfragments may be useful in serodiagnostic assays for canine leishmaniasis.

Animals↗
PubMed1996

Cytotoxicity, DNA binding, and reactivity against nucleosides of platinum (II) and (IV) spermine compounds.

We describe the synthesis and characterization of a [sperH4][PtCl4]2 salt and of five binuclear platinum (II) and (IV)-spermine compounds of formula [(PtCl2)2(sper)], cis-[(Pt(CH2(COO)2)2(sper)], cis-[(PtCBDCA)2(sper)], (CBDCA = 1,1'-cyclobutanedicarboxylate), cis-trans-cis[(PtCl2(OH)2)2(sper)], and cis-[(PtCl4)2(sper)], respectively. The 1H and 195Pt-NMR analysis of the complexes formed between these compounds and nucleosides indicated that the Pt centers show preferential binding to the N(7) of guanosine and adenosine residues, also being capable of forming bridged structures through the N(7) and N(1). The synthesized Pt-spermine compounds do not form complexes with cytidine residues at 37 degrees C. The circular dichroism, melting, and electrophoretic data of the compounds-DNA complexes show that the Pt(IV)-spermine complexes induce lower DNA conformational changes than their Pt(II) analogs. These results correlate with the IC50 values obtained against MDA-MB 468 and HL-60 human cancer cells which are higher than those of cis-DDP. The [sperH4][PtCl4]2 salt produces a high level of DNA modification and exhibits IC50 values lower than those of cis-DDP.

Antineoplastic Agents↗
PubMed1996

During canine viscero-cutaneous leishmaniasis the anti-Hsp70 antibodies are specifically elicited by the parasite protein.

A Leishmania infantum cDNA library was screened with sera from dogs with viscero-cutaneous leishmaniasis. Sequence analysis of a positive clone isolated from the library revealed that it coded for the carboxyl-terminal region of a member of the 70-kDa heat-shock protein family. The full-length sequence of the L. infantum hsp70 gene was determined after isolation of genomic clones. This protein shows a high degree of sequence conservation with the homologous protein from other organisms. To test its antigenicity a recombinant Hsp70 protein fused to the maltose-binding protein was produced in Escherichia coli using the expression vector pMAL-cRI. By FAST-ELISA assays it was observed that while the complete recombinant protein was recognized by 100% of the sera, the 20 carboxyl-terminal amino acids of the protein were only recognized by 30% of those sera. Thus, although a B-cell epitope must be present within the carboxyl terminal end of the protein other antigenic determinant(s) must reside out of this region. The analysis of the cross-reactivity with mouse Hsp70 by Western blotting strongly suggests that the anti-Hsp70 antibodies generated by infection with L. infantum are directed at specific determinants of the L. infantum Hsp70. Thus, our results indicate that anti-Hsp70 autoantibodies are not induced during Leishmania infection.

Amino Acid Sequence↗
PubMed1996

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↗
PubMed1996

Presence of antibodies in the aqueous humour and cerebrospinal fluid during Leishmania infections in dogs. Pathological features at the central nervous system.

In the present paper we show that in dogs, naturally infected with Leishmania infantum, the aqueous humour and the cerebrospinal fluid contain anti-Leishmania IgGs and that the specificity of antigen recognition of these fluids is similar to that of the sera. We also show that in the encephalon and cerebellum of these dogs there is a pathological sponge-like reaction accompanied by neuronal degeneration, mobilization of glial cells together with accumulation of amyloid deposits. The interstitial and intravascular deposition of IgGs and Leishmania antigens in choroid plexus suggest that in these animals there is a failure of the blood-cerebrospinal and ciliary bodies filtration barriers which may allow the transfer of anti-Leishmania IgGs from the blood stream to these fluids. We suggest that the failure of the blood-cerebrospinal barrier and the in situ concentration of anti-Leishmania IgGs and antigens in brain tissues may predispose to the pathological features detected in this compartment.

Animals↗
PubMed1996

Immunopathology of the uveitis in canine leishmaniasis.

Particular immunopathological features and their effects on the vascular permeability of different ocular structures were analysed in two dogs naturally infected by Leishmania infantum. The existence of specific anti-Leishmania immunoglobulin G (IgG) in the aqueous humour was confirmed by the ELISA technique. There was no correlation between antibody levels in the aqueous humour and the related serum. The histopathological study of the eyes showed the existence of lesions in various ocular structures. The ciliary processes, ciliary body, sclerocorneal limbus, iris and lacrimal duct showed intense inflammatory zones with lymphocyte infiltrates, plasmatic cells and macrophages with amastigote forms of Leishmania. In addition vasculitis with dilation and thrombi were also detected in both cases, with consequent oedema and hyalinization. The immunohistochemistry analysis revealed the presence of granular and diffuse IgG deposits in the ciliary body, ciliary processes, sclerocorneal limbus and iris. Furthermore, numerous thrombosed vessels were observed in the sclerocorneal zone and iris. Complement 3 (C3) fraction deposits were not present in the ocular structures. The present data suggest that the ocular lesions may have an immunopathological origin.

Animals↗
PubMed1996

Apoptosis: a mechanism of cell killing and lymphoid organ impairment during acute African swine fever virus infection.

Induction of programmed cell death has been described during infection with many different viruses. We have investigated the influence of African swine fever virus (ASFV) on apoptosis of different cell populations during in vitro and in vivo infection. We observed apoptosis in ASFV-infected monocyte/macrophage and peripheral blood mononuclear cell cultures. Apoptosis was demonstrated in these cells by DNA fragmentation, DNA staining and DNA-associated histone fraction detection assays. Flow cytometry analysis of infected cultures also showed morphological and functional alterations, including changes in the cell cycle and percentage of cell fractions stained with propidium iodide. After in vivo infection with three different virulent strains of ASFV, apoptosis of infected cells from the mononuclear phagocytic system and closely related elements from different tissues was observed. Additionally, infected pigs showed an intense degree of apoptosis of lymphocytes, which are not infected by the virus. In lymph nodes and other lymphoid organs, broad bands of apoptotic cells presented typical nuclear changes under light microscopy. The occurrence of DNA fragmentation was confirmed in these tissues using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling. These findings, together with the pathological observations in infected pigs of a depletion in cell populations in lymphoid organs, suggest that virus interference with programmed cell death plays a central role in pathogenesis of this disease, being responsible for lymphoid organ impairment in acute ASFV infection.

Acute Disease↗
PubMed1996

Specific serodiagnosis of human leishmaniasis with recombinant Leishmania P2 acidic ribosomal proteins.

The Leishmania P2 proteins have been analyzed as potential tools for the immunodiagnosis of human mucocutaneous and visceral leishmaniasis. Two recombinant Leishmania infantum proteins, rLIP2a and rLip2b, were used. The analysis indicated that the rLiP2a and rLiP2b proteins are recognized by 76% (16 of 21) and 42% (9 of 21), respectively, of sera from patients with mucocutaneous leishmaniasis and by 50% (5 of 10) and 40% (4 of 10), respectively, of sera from patients with visceral leishmaniasis. The Leishmania P2 proteins were engineered to have deletions of particular amino acids from the carboxyl-terminal region in order to avoid cross-reactivity with sera from patients with systemic lupus erythematosus and Chagas' disease, since it is known that this region is the main target of the autoantibodies present in sera from these patients. The results show that while the modified recombinant proteins rLiP2a-Q and rLiP2b-Q, in which the five carboxyl-terminal amino acids had been deleted, maintain the leishmaniasis-specific epitopes, they do not react with sera from patients with autoimmune disease and Chagas' disease. For this reason, and also because the sera from patients with tuberculosis and leprosy, diseases that have to be considered in a differential clinical diagnosis of infectious diseases, do not react with the rLiP2a-Q or rLiP2b-Q protein, we think that the engineered proteins may be considered specific tools for the immunodiagnosis of mucocutaneous and visceral leishmaniasis.

Adolescent↗
PubMed1996

Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus.

The infection of epithelia] swine testicle and intestinal porcine epithelial (IPEC-1) cell lines by adenovirus type 5 (Ad5) has been studied in vitro by using an Ad5-luciferase recombinant containing the firefly luciferase gene as a reporter. Porcine cell lines supported Ad5 replication, showing virus titers, kinetics of virus production, and luciferase expression levels similar to those obtained in human 293 cells, which constitutively express the 5'-end 11% of the Ad5 genome. The tropism of Ad5-based vectors in swine and its ability to induce an efficient immune response against heterologous antigens expressed by foreign genes inserted in these vectors has been determined. Ad5 vectors replicate and express heterologous antigens in porcine lungs and mediastinal and mesenteric lymph nodes. Significant levels of heterologous antigen expression were also demonstrated in the small intestine (jejunum and ileum), but Ad5 replication in this organ was very poor, suggesting that Ad vectors undergo an abortive replication in the porcine small intestine. The tissues infected by Ad5 were dependent on the inoculation route. The oronasal route appeared to be best for inoculation of bronchus-associated lymphoid tissue infection, while the intraperitoneal route was best for gut-associated lymphoid tissue infection. Epithelial cells of bronchioles, macrophages, type II pneumocytes, and follicular dendritic cells were identified as targets for Ad5, while epithelial cells of the intestine were not infected by Ad5. Viruses with a deletion from 79.5 to 84.8 map units in the E3 region, with or without heterologous inserted genes, replicated to lower levels in porcine tissues than did wild-type Ad5. It was also shown that an Ad5 recombinant expressing the four antigenic sites (A, B, C, and D) of transmissible gastroenteritis coronavirus (TGEV) spike protein induced in swine immune responses which neutralized TGEV infectivity. In addition, porcine serum from Ad-TGEV-immune animals provide passive protection when mixed with fully virulent TGEV and orally administered to highly susceptible newborn piglets. These results taken together indicate that swine may be a good animal model for human Ad5 lung infection to aid in the evaluation of candidate adenovirus vaccines and that Ad5 may be suitable as a recombinant viral vaccine or for other applications in swine.

Adenoviruses, Human↗
PubMed1996

Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization.

African swine fever virus induces in convalescent pigs antibodies that neutralized the virus before and after binding to susceptible cells, inhibiting both virus attachment and internalization. A further analysis of the neutralization mechanisms mediated by the different viral proteins showed that antibodies to proteins p72 and p54 are involved in the inhibition of a first step of the replication cycle related to virus attachment, while antibodies to protein p30 are implicated in the inhibition of virus internalization.

African Swine Fever↗
PubMed1996

[Clinical relevance of gram-negative bacteria having inducible chromosomic beta-lactamase at an intensive care unit].

BACKGROUND: The aim of the study was to determine the frequency of third-generation cephalosporins and aztreonam resistance in gram-negative bacteria with inducible chromosomal beta-lactamase (beta Lac-ind) after beta-lactam therapy in the medical-surgical intensive care unit (ICU) at a university-affiliated hospital. PATIENTS AND METHODS: We studied 34 infections in 29 patients admitted to the ICU. All were infected by strains with beta Lac-ind and all were treated with beta-lactam antibiotics. Susceptibility was determined by disc-diffusion. The beta-lactamase activity of those strains showing constitutive beta-lactamase overproduction were characterized by isoelectrofocusing. When this derepression occurred during the therapy, the strains were compared by genomic macrorestriction (PGFE). RESULTS: In 29 out of 34 infections the initial strains was susceptible. In 11 cases, the culture were not negativized in spite of their susceptible pattern. In 4 cases there was derepression during therapy. In 5 cases the initial strains were derepressed. The microorganisms isolated more frequently were Pseudomonas aeruginosa (22 cases) and Enterobacter cloacae (5 cases). The beta-lactamase activity detected correspond well with a betaLac-ind. In those cases with derepression during therapy, the initial susceptible strain and the resistant strain were identical by PGFE.

Adolescent↗
PubMed1996

[Applications of the polymerase chain reaction (PCR) to the diagnosis of central nervous system infections].

The utility of polymerase chain reaction (PCR) is described for the diagnosis in three patients suffering from central nervous system infections, tuberculous meningitis, herpetic encephalitis and cerebral toxoplasmosis. PCR was performed in the cerebrospinal fluid after processing the specimen by two methods, proteinase K digestion and phenol extraction of DNA. Amplification was realized using primers previously described that amplify specific DNA fragments of each microorganisms (insertion sequence IS6110 of Mycobacterium tuberculosis, B1 gene of Toxoplasma gondii, and DNA polymerase gene of Herpes simplex virus). In all three cases, PCR was positive after amplification of the specimen extracted with proteinase K, as well as when a complete DNA extraction with phenol was realized. In all cases a band of amplified products was observed in agarose gels. In conclusion, in all three patients described, PCR would had allowed the diagnosis in seven hours, and PCR should be consider a rapid sensitive and relatively simple method.

AIDS-Related Opportunistic Infections↗