María del Carmen Thomas

Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide

BACKGROUND Celiac disease is an immune-mediated enteropathy caused by the ingestion of gluten, a protein fraction found in certain cereals. Immunotoxic gluten peptides that are recalcitrant to degradation of digestive enzymes appear to trigger celiac syndromes. A 33-mer peptide from alpha-2 gliadin has been identified as a principal contributor to gluten immunotoxicity. A gluten-free diet is the usual first therapy for celiac disease patients; therefore, the characterization and quantification of the toxic portion of the gluten in foodstuffs is crucial to avoid celiac damage. OBJECTIVE We aimed to develop immunologic assays as a novel food analysis tool for measuring cereal fractions that are immunotoxic to celiac disease patients. DESIGN The design focused on the production of monoclonal antibodies against the gliadin 33-mer peptide and the development of enzyme-linked immunosorbent assays (ELISAs) and Western blot analysis with the use of novel antibodies. RESULTS A sandwich ELISA method showed a detection limit for wheat, barley, and rye of <1 ppm prolamine. However, the method required a sample that was > or =1 order of magnitude greater for the detection of low-toxic oats, and there was no signal with the safe cereals maize and rice. A competitive ELISA method was also developed for detection of the toxic peptide in hydrolyzed food, which had a detection limit of <0.5 ppm gliadin. CONCLUSIONS Both ELISAs designed for use with the toxic gliadin 33-mer peptide suggested a high correlation between the presence of the peptide and the amount of cereal that was toxic to celiac disease patients. The sensitivity was significantly higher than that of equivalent methods recognizing other gluten epitopes.

Immunogenicity of HSP-70, KMP-11 and PFR-2 leishmanial antigens in the experimental model of canine visceral leishmaniasis

Zoonotic visceral leishmaniasis (ZVL) is a parasitic disease caused by Leishmania infantum/L. chagasi that is emerging as an important medical and veterinary problem. Dogs are the domestic reservoir for this parasite and, therefore, the main target for controlling the transmission to humans. In the present work, we have evaluated the immunogenicity of the Leishmania infantum heat shock protein (HSP)-70, paraflagellar rod protein (PFR)-2 and kinetoplastida membrane protein (KMP)-11 recombinant proteins in dogs experimentally infected with the parasite. We have shown that peripheral blood mononuclear cells (PBMC) from experimentally infected dogs proliferated in response to these recombinant antigens and against the soluble leishmanial antigen (SLA). We have also quantified the mRNA expression level of the cytokines induced in PBMC upon stimulation with the HSP-70, PFR-2 and KMP-11 proteins. These recombinant proteins induced an up-regulation of IFN-gamma. HSP-70 and PFR-2 also produced an increase of the TNF-alpha transcripts abundance. No measurable induction of IL-10 was observed and low levels of IL-4 mRNA were produced in response to the three mentioned recombinant antigens. Serum levels of specific antibodies against HSP-70, PFR-2 and KMP-11 recombinant proteins were also determined in these animals. Our study showed that HSP-70, KMP-11 and PFR-2 proteins are recognized by infected canines. Furthermore, these antigens produce a Th1-type immune response, suggesting that they may be involved in protection. The identification as vaccine candidates of Leishmania antigens that elicit appropriate immune responses in the canine model is a key step in the rational approach to generate a vaccine for canine visceral leishmaniasis.

Differential CD86 and CD40 co-stimulatory molecules and cytokine expression pattern induced by Trypanosoma cruzi in APCs from resistant or susceptible mice.

Differential aspects of the host immune response generated by Trypanosoma cruzi infection were examined in two different mouse strains, BALB/c (haplotype H2‐Kd) which does not overcome the acute phase of the infection and C57BL/6 (haplotype H2‐Kb) which survives to the acute phase. After infection an increase in CD3+ T cells was observed in both mouse strains in the peritoneal cavity. However, while the CD3+ T cells from the BALB/c mice showed an increase in the IL‐4 cytokine expression level, the same type of cells from the C57BL/6 mice showed an increase in IFN‐γ expression. In addition, only the macrophages from the C57BL/6 mice were activated secreting IL‐12 and TNF‐α and producing, moreover, high levels of nitrites. It was observed that also after parasite infection the expression of macrophage and dendritic cells CD40 and CD86 co‐stimulation molecules from the spleen were diminished in BALB/c but not in C57BL/6 mice. In correlation with this observation the macrophages from the spleen of infected BALB/c mice secreted lower concentrations of nitrites than the C57BL/6 mouse cells. Also, the spleen dendritic cells from infected BALB/c mice had a small potential to present alloantigens in contrast to that observed in the infected C57BL/6 mouse cells.

Trypanosoma cruzi heat-shock protein-70 kDa,alone or fused to the parasite KMP11 antigen, induces functional maturation of murine dendritic cells.

We analyse the effect of Trypanosoma cruzi heat‐shock protein‐70 (HSP70) on the maturation of murine dendritic cells (DC) generated from bone marrow precursor cells. The results obtained show that HSP70, both alone and fused to the KMP11 antigen, as well as a HSP70 fragment, is capable of maturing murine DC. Mature DC have enhanced expression of IL12, TNF‐α cytokines, costimulation molecules and activation markers, showing a clear increase in the allostimulatory capacity. These findings suggest that T. cruzi HSP70 may be a very useful vehicle for developing DC‐based immunoprophylaxis and therapy against infections.

The L1Tc, long interspersed nucleotide element from Trypanosoma cruzi, encodes a protein with 3'-phosphatase and 3'-phosphodiesterase enzymatic activities.

The presence of a long interspersed nucleotide element, named L1Tc, which is actively transcribed in the parasiteTrypanosoma cruzi, has been recently described. The open reading frame 1 of this element encodes the NL1Tc protein, which has apurinic/apyrimidinic endonuclease activity and is probably implicated in the first stage of the transposition of the element. In the present paper we show that NL1Tc effectively removes 3′-blocking groups (3′-phosphate and 3′-phosphoglycolate) from damaged DNA substrates. Thus, both 3′-phosphatase and 3′-phosphodiesterase activities are present in NL1Tc. We propose that these enzymatic activities would allow the 3′-blocking ends to function as targets for the insertion of L1Tc element, in addition to the apurinic/apyrimidinic sites previously described. The potential biological function of the NL1Tc protein has also been evidenced by its ability to repair the DNA damage induced by the methyl methanesulfonate alkylating or oxidative agents such as hydrogen peroxide and t-butyl hydroperoxide inEscherichia coli (xth andxth, nfo) mutants.

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 3200 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.

Characterization of reverse transcriptase activity of the L1Tc retroelement from Trypanosoma cruzi

The recombinant protein RTL1Tc, encoded by the non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi, has been shown to have reverse transcriptase (RT) activity using poly(rA)/oligo(dT) and poly(rC)/oligo(dG) homopolymers as template/primers. The optimal RT activity was detected at a concentration of 5 mM Mg2+, pH 8 and between 28 and 37%°C. Site-directed mutagenesis in the RT catalytic site proved that substitution of aspartic acid 313 for isoleucine (RTD313IL1Tc) practically abolishes the RT activity of the RTL1Tc protein. RT-polymerase chain reaction assays revealed that the RTL1Tc protein has the ability to use both homologous and heterologous RNA templates. Also, it is shown that the RTL1Tc protein is capable of synthesizing complementary DNA molecules by consecutive switching of the oligo molecule, which the protein uses as a template. This template switching may be involved in the retroelement integration process.

Natural CD4 + T-cell responses against Trypanosoma cruzi KMP-11 protein in chronic chagasic patients

Trypanosoma cruzi kinetoplastid membrane protein‐11 (KMP‐11) is able to induce protective immunity in mice. In humans, T‐cell immunity during Chagas disease has been documented using parasite antigens allowing the identification of specific CD8+ T cells. However, little is known about the CD4+ T‐cell response during the evolution of the disease. In this paper, the induction of a natural CD4+ T‐cell response against the KMP‐11 protein in T. cruzi infected humans was studied to assess whether this parasite‐derived protein could be processed, presented and detected as a major histocompatibility complex class II restricted epitope. The results show that helper T cells from 5 out of 13 chagasic patients specifically produced interferon‐γ after exposure to the KMP‐11 antigen, whereas healthy donors and non‐chagasic cardiopathic patients did not respond. This is the first description of T. cruzi KMP‐11 protein recognition by CD4+ T cells in chronic chagasic patients.

The endonuclease NL1Tc encoded by the LINE L1Tc from Trypanosoma cruzi protects parasites from daunorubicin DNA damage

In the present paper we show that the overexpression of the NL1Tc protein, encoded by the L1Tc non-LTR retrotransposon from Trypanosoma cruzi, led to a reduction of about 60% of DNA damage caused by daunorubicin treatment. This repair effect is not observed in transfected parasites overexpressing the NL1Tc mutated in the aspartic acid located in the active site of the enzyme. In addition, NL1Tc overexpression protects the parasite from the negative effect that daunorubicin has on parasites growth rate. Thus, parasites overexpressing NL1Tc show, after treatment with 4 microM of daunorubicin, growth rate two to three times higher than the growth rate observed in treated control parasites transformed with the empty vector or overexpressing the mutated NL1Tc. Likewise, parasites overexpressing the NL1Tc protein and irradiated with a single dose of gamma-radiation (6 or 9 Gy) show higher growth rates than the parasites overexpressing the mutated NL1Tc or the control transfected parasites.

Molecular characterization of the histone H2A gene from the parasite Trypanosoma rangeli.

Abstract The sequence, genomic organization, and transcription of the gene encoding the H2A histone protein of the protozoan parasite Trypanosoma rangeli is described in this paper. The locus encoding the T. rangeli H2A protein is formed by at least 11 gene units measuring 790 nucleotides in length, organized in tandem, and located in a single chromosome of approximately 1.9 Mb. The gene units actively transcribe only one size class of mRNA measuring 0.7 kb in length. The T. rangeli H2A protein contains in the amino-terminal the AGLXFPV motif, which is conserved in a broad range of H2A proteins, and the RSAK motif, which is implicated in repression of the histones basal transcription in yeast. The carboxyl-terminal of the protein contains a two-lysine residue described as the ubiquitin binding site and the histidine residue implicated in DNA binding.