Antiparasitic Treatment Induces an Improved CD8+ T Cell Response in Chronic Chagasic Patients.

Chagas disease is a chronic infection caused by Trypanosoma cruzi, an intracellular protozoan parasite. Chronic chagasic patients (CCPs) have dysfunctional CD8+ T cells that are characterized by impaired cytokine production, high coexpression of inhibitory receptors, and advanced cellular differentiation. Most patients diagnosed in the chronic phase of Chagas disease already exhibit heart involvement, and there is no vaccination that protects against the disease. Antiparasitic treatment is controversial as to its indication for this stage of the disease. There is a lack of biological markers to evaluate the effectiveness of antiparasitic treatment, and little is known about the effect of the treatment on CD8+ T cells. Thus, the aim of the current study was to analyze the early effects of antiparasitic treatment on CD8+ T cells from CCPs with asymptomatic clinical forms of disease. To evaluate the CD8+ T cell subsets, expression of inhibitory receptors, and functionality of T cells in CCPs, PBMCs were isolated. The results showed that treatment of CCPs with the asymptomatic form of the disease induces an increase in the frequency of CD8+ central memory T cells and terminal effector T cells, a decrease in the coexpression of inhibitory receptors, an improved Ag-specific CD8+ T cell response exhibited by the individual production of IFN-γ or IL-2, and a multifunctional CD8+ T cell profile of up to four functions (IFN-γ+IL-2+Perforin+Granzyme B+). These findings suggest that, in CCPs, antiparasitic treatment improved the quality of Ag-specific CD8+ T cell responses associated with a decrease in inhibitory receptor coexpression, which could serve as biomarkers for monitoring the effectiveness of antiparasitic treatment.

Biology of Trypanosoma cruzi Retrotransposons: From an Enzymatic to a Structural Point of View.

INTRODUCTION: An important portion of the Trypanosoma cruzi genome is composed of mobile genetic elements, which are interspersed with genes on all chromosomes. The L1Tc non-LTR retrotransposon and its truncated version NARTc are the most highly represented and best studied of these elements. L1Tc is actively transcribed in all three forms of the Trypanosoma parasite and encodes the proteins that enable it to autonomously mobilize. This mini review discusses the enzymatic properties of L1Tc that enable its mobilization and possibly the mobilization of other non-autonomous retrotransposons in Trypanosoma. We also briefly review the Hepatitis Delta Virus-like autocatalytic and 2A self-cleaving viral-like sequences contained in L1Tc that regulate post-transcriptional properties such as relative protein abundance and mRNA stability. Special emphasis is placed on the Pr77 dual system, which is based on the RNA pol II-dependent internal promoter of L1Tc and NARTc and the HDV-like ribozyme activity encoded by the first 77 nucleotides of the element's DNA and RNA. The high degree of conservation of the Pr77 sequence, referred to as the "Pr77-hallmark", among different trypanosomatid retroelements suggests that these mobile elements are responsible for the distribution of regulatory sequences within the genome they inhabit. CONCLUSION: We also discuss how the involvement of L1Tc and NARTc in the gene regulatory processes of these parasites could justify their domestication and long-term coexistence in these ancient organisms.

Inhibitory Receptor Expression on CD8+ T Cells Is Linked to Functional Responses against Trypanosoma cruzi Antigens in Chronic Chagasic Patients.

In mammals, chronic diseases resulting from infectious agents have been associated with functional T cell response deficiency, a high frequency of terminally differentiated T cells, the presence of monofunctional Ag-specific T cells, and increased expression of inhibitory receptors. Similar to other chronic diseases, the progressive loss of certain functional activities during Trypanosoma cruzi infection might result in the inability to control replication of this parasite. To examine this hypothesis, we evaluated the differentiation and cell effector function of CD8(+) T cells and characterized the expression of inhibitory receptors and the presence of the parasite in the bloodstream of chagasic patients. The results showed that patients at an advanced severe disease stage had a higher frequency of terminally differentiated CD8(+) T cells than patients at an early stage of the disease. A monofunctional CD8(+) T cell response was observed in patients at an advanced stage, whereas the coexpression of markers that perform three and four functions in response to parasite Ags was observed in patients at a less severe disease stage. The frequency of CD8(+) T cells producing granzyme B and perforin and those expressing inhibitory receptors was higher in symptomatic patients than in asymptomatic patients. Taken together, these findings suggest that during the course of Chagas disease, CD8(+) T cells undergo a gradual loss of function characterized by impaired cytokine production, the presence of advanced differentiation, and increased inhibitory receptor coexpression.

Identification of a type I nitroreductase gene in non-virulent Trypanosoma rangeli.

Trypanosomatid type I nitroreductases (NTRs), i.e., mitochondrial enzymes that metabolise nitroaromatic pro-drugs, are essential for parasite growth, infection, and survival. Here, a type I NTR of non-virulent protozoan Trypanosoma rangeli is described and compared to those of other trypanosomatids. The NTR gene was isolated from KP1(+) and KP1(-) strains, and its corresponding transcript and 5' untranslated region (5'UTR) were determined. Bioinformatics analyses and nitro-drug activation assays were also performed. The results indicated that the type I NTR gene is present in both KP1(-) and KP1(+) strains, with 98% identity. However, the predicted subcellular localisation of the protein differed among the strains (predicted as mitochondrial in the KP1(+) strain). Comparisons of the domains and 3D structures of the NTRs with those of orthologs demonstrated that the nitroreductase domain of T. rangeli NTR is conserved across all the strains, including the residues involved in the interaction with the FMN cofactor and in the tertiary structure characteristics of this oxidoreductase protein family. mRNA processing and expression were also observed. In addition, T. rangeli was shown to be sensitive to benznidazole and nifurtimox in a concentration-dependent manner. In summary, T. rangeli appears to have a newly discovered functional type I NTR.

Altering the motility of Trypanosoma cruzi with rabbit polyclonal anti-peptide antibodies reduces infection to susceptible mammalian cells.

Trypanosoma cruzi's trypomastigotes are highly active and their incessant motility seems to be important for mammalian host cell infection. The kinetoplastid membrane protein-11 (KMP-11) is a protein expressed in all parasite stages, which induces a cellular and humoral immune response in the infected host, and is hypothesized to participate in the parasite's motility. An N-terminal peptide from KMP-11, termed K1 or TcTLE, induced polyclonal antibodies that inhibit parasitic invasion of Vero cells. The goal of this study was to evaluate the motility and infectivity of T. cruzi when exposed to polyclonal anti-TcTLE antibodies. Rabbits were immunized with TcTLE peptide along with FIS peptide as an immunomodulator. ELISA assay results showed that post-immunization sera contained high titers of polyclonal anti-TcTLE antibodies, which were also reactive against the native KMP-11 protein and live parasites as detected by immunofluorescence and flow cytometry assays. Trypomastigotes of T. cruzi were incubated with pre- or post-immunization sera, and infectivity to human astrocytes was assessed by Giemsa staining/light microscope and flow cytometry using carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled parasites. T. cruzi infection in astrocytes decreased approximately by 30% upon incubation with post-immunization sera compared with pre-immunization sera. Furthermore, trypomastigotes were recorded by video microscopy and the parasite's flagellar speed was calculated by tracking the flagella. Trypomastigotes exposed to post-immunization sera had qualitative alterations in motility and significantly slower flagella (45.5 µm/s), compared with those exposed to pre-immunization sera (69.2 µm/s). In summary, polyclonal anti-TcTLE serum significantly reduced the parasite's flagellar speed and cell infectivity. These findings support that KMP-11 could be important for parasite motility, and that by targeting its N-terminal peptide infectivity can be reduced.

Risk factors and primary prevention of congenital Chagas disease in a nonendemic country.

BACKGROUND: In this longitudinal cohort study we evaluated the congenital transmission of Chagas disease (CD) in a nonendemic area. The aim of this work was to analyze the predictive value of a Trypanosoma cruzi-positive polymerase chain reaction (PCR) result in pregnant women for the diagnosis of vertical transmission and to evaluate the use of PCR as a tool for early detection of infection. METHODS: The offspring of 59 seropositive pregnant mothers were followed up. The parasitological status of mothers was studied by PCR in a total of 64 pregnancies; 10 of these women had received treatment before pregnancy. Sixty-five infants (including a pair of twins) were monitored at 0, 6, 9, and 12 months of age by PCR and serology. In cases of congenital transmission, hemoculture and parasite lineage typing were performed. RESULTS: Nine infants had acquired CD congenitally. This represents a transmission rate of 13.8% among seropositive mothers (9 infected newborns of 65 total live births). All infants were infected with T. cruzi discrete typing unit V strain. A statistically significant correlation was found between T. cruzi vertical transmission and a positive PCR result during pregnancy (31%; 9 infected newborns in 29 live births). No infected infants were detected among 10 mothers who were treated before they became pregnant, compared with 16.4% (9 of 55 live births) among untreated mothers. CONCLUSIONS: PCR is a useful tool for the detection of congenital CD, and the treatment of infected women of childbearing age seems to be useful for preventing vertical transmission.

Sequence polymorphism in the Trypanosoma rangeli HSP70 coding genes allows typing of the parasite KP1(+) and KP1(-) groups.

The genes encoding the Trypanosoma rangeli heat shock protein 70kDa were sequenced and their genomic organization determined. This human parasite has medical relevance as it shares antigens, hosts and geographical regions with the etiological agent of Chagas' disease, Trypanosoma cruzi. The T. rangeli HSP70 genes are highly conserved regarding their tandem organization, and deduced amino acid sequences among T. rangeli KP1(+) and KP1(-) groups and other trypanosomatids. Nevertheless, a variable number of the immunogenic GMPG motif was observed among HSP70 copies within the same T. rangeli isolate and among different isolates. Interestingly, a polymorphism at nucleotide level affecting the SphI restriction site allowed the differentiation of KP1(-) and KP1(+) groups.

Identification of an hepatitis delta virus-like ribozyme at the mRNA 5'-end of the L1Tc retrotransposon from Trypanosoma cruzi.

L1Tc is a non-LTR LINE element from Trypanosoma cruzi that encodes its transposition machinery and bears an internal promoter. Herewith, we report the identification of an in vitro active hepatitis delta virus-like ribozyme located in the first 77 nt at the 5'-end of the L1Tc mRNA (L1TcRz). The data presented show that L1TcRz has a co-transcriptional function. Using gel-purified uncleaved RNA transcripts, the data presented indicate that the kinetics of the self-cleaving, in a magnesium-dependent reaction, fits to a two-phase decay curve. The cleavage point identified by primer extension takes place at +1 position of the element. The hydroxyl nature of the 5'-end of the 3'-fragment generated by the cleavage activity of L1TcRz was confirmed. Since we have previously described that the 77-nt long fragment located at the 5'-end of L1Tc has promoter activity, the existence of a ribozyme in L1Tc makes this element to be the first described non-LTR retroelement that has an internal promoter-ribozyme dual function. The L1Tc nucleotides located downstream of the ribozyme catalytic motif appear to inhibit its activity. This inhibition may be influenced by the existence of a specific L1Tc RNA conformation that is recognized by RNase P.

Molecular characterization of the short interspersed repetitive element SIRE in the six discrete typing units (DTUs) of Trypanosoma cruzi.

Repetitive sequences constitute an important proportion of the Trypanosoma cruzi genome; hence, they have been used as molecular markers and as amplification targets to identify the parasite presence via PCR. In this study, a molecular characterization of the SIRE repetitive element was performed in the six discrete typing units (DTUs) of T. cruzi. The results evidenced that this element, located in multiple chromosomes, was interspersed in the genome of all DTUs of the parasite. The presence of several motifs implicated in element insertion, duplication, and functionality suggests that SIRE could be an active element in the parasite genome. Of interest, there were SIRE specific Alu I fragments that allowed to discriminate DTU I from the others DTUs. Moreover, an UPGMA phenetic tree constructed from fragment sharing Southern blot data showed that T. cruzi I isolates conform a cluster separated from the T. cruzi II-VI isolates. When the relative number of SIRE copies was determined, a variation from 105 to 2,000 copies per haploid genome was observed among the different isolates without kept a DTU-relationship. In all, these findings suggest that SIRE sequence is a good target for parasite DNA amplification.

Short-term follow-up of chagasic patients after benzonidazole treatment using multiple serological markers.

BACKGROUND: Conventional serological tests, using total soluble proteins or a cocktail of recombinant proteins from T. cruzi as antigens, are highly sensitive for Chagas disease diagnosis. This type of tests, however, does not seem to be reliable tools for short- and medium-term monitoring of the evolution of patients after antiparasitic treatment. The aim of the present study was to search for immunological markers that could be altered in the sera from Chagas disease patients after benznidazole treatment, and therefore have a potential predictive diagnostic value. METHODS: We analyzed the reactivity of sera from chagasic patients during different clinical phases of the disease against a series of immunodominant antigens, known as KMP11, PFR2, HSP70 and Tgp63. The reactivity of the sera from 46 adult Chronic Chagas disease patients living in a non-endemic country without vector transmission of T. cruzi (15 patients in the indeterminate stage, 16 in the cardiomiopathy stage and 16 in the digestive stage) and 22 control sera from non-infected subjects was analyzed. We also analyzed the response dynamics of sera from those patients who had been treated with benznidazole. RESULTS: Regardless of the stage of the sickness, the sera from chagasic patients reacted against KMP11, HSP70, PFR2 and Tgp63 recombinant proteins with statistical significance relative to the reactivity against the same antigens by the sera from healthy donors, patients with autoimmune diseases or patients suffering from tuberculosis, leprosy or malaria. Shortly after benznidazole treatment, a statistically significant decrease in reactivity against KMP11, HSP70 and PFR2 was observed (six or nine month). It was also observed that, following benznidazole treatment, the differential reactivity against these antigens co-relates with the clinical status of the patients. CONCLUSIONS: The recombinant antigens KMP11, PFR2, Tgp63 and HSP70 are recognized by Chagas disease patients' sera at any clinical stage of the disease. Shortly after benznidazole treatment, a drop in reactivity against three of these antigens is produced in an antigen-specific manner. Most likely, analysis of the reactivity against these recombinant antigens may be useful for monitoring the effectiveness of benznidazole treatment.

Nucleic-acid-binding properties of the C2-L1Tc nucleic acid chaperone encoded by L1Tc retrotransposon.

It has been reported previously that the C2-L1Tc protein located in the Trypanosoma cruzi LINE (long interspersed nuclear element) L1Tc 3' terminal end has NAC (nucleic acid chaperone) activity, an essential activity for retrotransposition of LINE-1. The C2-L1Tc protein contains two cysteine motifs of a C2H2 type, similar to those present in TFIIIA (transcription factor IIIA). The cysteine motifs are flanked by positively charged amino acid regions. The results of the present study show that the C2-L1Tc recombinant protein has at least a 16-fold higher affinity for single-stranded than for double-stranded nucleic acids, and that it exhibits a clear preference for RNA binding over DNA. The C2-L1Tc binding profile (to RNA and DNA) corresponds to a non-co-operative-binding model. The zinc fingers present in C2-L1Tc have a different binding affinity to nucleic acid molecules and also different NAC activity. The RRR and RRRKEK [NLS (nuclear localization sequence)] sequences, as well as the C2H2 zinc finger located immediately downstream of these basic stretches are the main motifs responsible for the strong affinity of C2-L1Tc to RNA. These domains also contribute to bind single- and double-stranded DNA and have a duplex-stabilizing effect. However, the peptide containing the zinc finger situated towards the C-terminal end of C2-L1Tc protein has a slight destabilization effect on a mismatched DNA duplex and shows a strong preference for single-stranded nucleic acids, such as C2-L1Tc. These results provide further insight into the essential properties of the C2-L1Tc protein as a NAC.

Evaluation of the circadian rhythm of anti-Leishmania IgG2 and IgA antibodies in serum and saliva of dogs with clinical leishmaniosis.

In this study, the circadian rhythm of IgG2 and IgA specific antibodies in serum and saliva samples of 6 dogs experimentally infected with Leishmania infantum was assessed. Sampling was performed at 8.00, 12.00, 16.00, 20.00, and 00.00 h on two consecutive days. Anti-Leishmania antibody levels in serum were expressed without any correction, whereas in saliva were shown in different ways: without any correction, adjusted by protein concentration and corrected by the salivary flow rate. No significant differences in anti-Leishmania IgG2 antibody levels in serum and saliva samples with or without correction were found. Significant differences were found when anti-Leishmania IgA levels were corrected by the salivary flow rate. In addition, a greater intra-individual variation of antibody levels was observed in saliva than in serum. However, this variation did not modify the serological status of the dogs. Therefore, it could be concluded that there is no circadian rhythm in serum and saliva samples and sampling can be performed at any time of the day.

Detection of Leishmania infantum DNA by real-time PCR in saliva of dogs.

This study developed a real-time quantitative PCR (qPCR) assay to detect L. infantum kinetoplast DNA (kDNA) in canine saliva. The qPCR showed an efficiency of 93.8%, a coefficient of correlation of 0.996 and a detection limit of 0.5 fg/reaction (0.005 parasites), although it detected until 0.25 fg/reaction (0.0025 parasites). When samples from 12 dogs experimentally infected with L. infantum were collected, L. infantum kDNA was detected at 16-weeks post-infection (wpi) in 41.7% and 91.7% of saliva and bone marrow samples, respectively, and at 47-wpi in 75% of both samples. L. infantum kDNA can be detected by qPCR in canine saliva, with lower sensitivity in the early stages of infection and a lower parasite load estimation compared to bone marrow. However, saliva had similar sensitivities to bone marrow in the later stages of the infection and could be used to detect L. infantum kDNA being aware of its limitations.

Serum proteome of dogs at subclinical and clinical onset of canine leishmaniosis.

The objective of this study was to identify changes in serum proteome in dogs that may occur after an experimental infection at subclinical and clinical stages of canine leishmaniosis (CanL). For this purpose, canine pre- and post-infection with Leishmania infantum serum proteomes in the same dogs were analysed by a high-throughput label-based quantitative LC-MS/MS proteomic approach. A total of 169 proteins were identified, and 74 of them including complement C8 alpha chain, adiponectin, transferrin, sphingomyelin phosphodiesterase acid-like 3A and immunoglobulins showed different modulation between the different stages of CanL. These proteins could be considered as potential serum biomarkers of early diagnostic or disease progression in CanL. Additionally, biological pathways modulated during CanL such as blood coagulation or gonadotropin-releasing hormone receptor were revealed, which could help to understand the pathological mechanisms of the disease.

The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts.

L1Tc is the best represented autonomous LINE of the Trypanosoma cruzi genome, throughout which several functional copies may exist. In this study, we show that the first 77 bp of L1Tc (Pr77) (also present in the T. cruzi non-autonomous retrotransposon NARTc, in the Trypanosoma brucei RIME/ingi elements, and in the T. cruzi, T. brucei and Leishmania major degenerate L1Tc/ingi-related elements [DIREs]) behave as a promoter element that activates gene transcription. The transcription rate promoted by Pr77 is 10-14-fold higher than that mediated by sequences located upstream from the T. cruzi tandemly repeated genes KMP11 and the GAPDH. The Pr77 promoter-derived mRNAs initiate at nucleotide +1 of L1Tc, are unspliced and translated. L1Tc transcripts show a moderate half life and are RNA pol II dependent. The presence of an internal promoter at the 5' end of L1Tc favors the production of full-length L1Tc RNAs and reinforces the hypothesis that this mobile element may be naturally autonomous in its transposition.

One-year follow-up of anti-Leishmania antibody concentrations in serum and saliva from experimentally infected dogs.

The quantification of anti-Leishmania antibodies in serum and saliva by a time-resolved immunofluorometric assay is useful for the diagnosis and treatment monitoring of dogs with clinical leishmaniasis. We compared the kinetics of anti-Leishmania IgG2 and IgA antibodies in serum and saliva from 11 Beagle dogs experimentally infected with Leishmania infantum. Most dogs showed detectable concentrations of anti-Leishmania IgG2 earlier in serum (between 3 and 4 months p.i.) than in saliva (between 4 and 6 months p.i.). Overall, a high correlation between concentrations of anti-Leishmania IgG2 in serum and saliva (r = 0.853; P < 0.0001) was observed. The quantification of anti-Leishmania IgA showed less diagnostic value than IgG2, since detectable amounts of IgA were not observed in the saliva of four dogs and in the serum of one dog. In addition, a very low correlation between anti-Leishmania IgA in serum and saliva (r = 0.289; P < 0.001) was observed. Our results indicate that the antibodies against L. infantum in saliva appear approximately 1 month later than in serum, and suggest that there is a threshold for the passing of immunoglobulins from serum to saliva in dogs. These facts should be taken into consideration for a proper interpretation of saliva assays for quantification of antibodies.

Changes in saliva of dogs with canine leishmaniosis: A proteomic approach.

In the present study, a quantitative proteomic approach to study changes in saliva proteins associated with canine leishmaniosis (CanL) was performed. For this, canine salivary proteins were analysed and compared between dogs before (T0) and after (T1) experimental infection with Leishmania infantum by high-throughput label-based quantitative LC-MS/MS proteomic approach and bioinformatic analysis of the in silico inferred interactome protein network was created from the initial list of differential proteins. More than 2000 proteins were identified, and of the 90 differentially expressed proteins between T0 and T1, 12 were down-regulated with log2 fold change lower than -0.5849, and 19 were up-regulated with log2 fold change greater than 0.5849. This study provides evidence of changes in salivary proteome that can occur in canine leishmaniosis and revealed biological pathways in saliva modulated in canine leishmaniosis with potential for further targeted research.

The SIDER2 elements, interspersed repeated sequences that populate the Leishmania genomes, constitute subfamilies showing chromosomal proximity relationship.

BACKGROUND: Protozoan parasites of the genus Leishmania are causative agents of a diverse spectrum of human diseases collectively known as leishmaniasis. These eukaryotic pathogens that diverged early from the main eukaryotic lineage possess a number of unusual genomic, molecular and biochemical features. The completion of the genome projects for three Leishmania species has generated invaluable information enabling a direct analysis of genome structure and organization. RESULTS: By using DNA macroarrays, made with Leishmania infantum genomic clones and hybridized with total DNA from the parasite, we identified a clone containing a repeated sequence. An analysis of the recently completed genome sequence of L. infantum, using this repeated sequence as bait, led to the identification of a new class of repeated elements that are interspersed along the different L. infantum chromosomes. These elements turned out to be homologues of SIDER2 sequences, which were recently identified in the Leishmania major genome; thus, we adopted this nomenclature for the Leishmania elements described herein. Since SIDER2 elements are very heterogeneous in sequence, their precise identification is rather laborious. We have characterized 54 LiSIDER2 elements in chromosome 32 and 27 ones in chromosome 20. The mean size for these elements is 550 bp and their sequence is G+C rich (mean value of 66.5%). On the basis of sequence similarity, these elements can be grouped in subfamilies that show a remarkable relationship of proximity, i.e. SIDER2s of a given subfamily locate close in a chromosomal region without intercalating elements. For comparative purposes, we have identified the SIDER2 elements existing in L. major and Leishmania braziliensis chromosomes 32. While SIDER2 elements are highly conserved both in number and location between L. infantum and L. major, no such conservation exists when comparing with SIDER2s in L. braziliensis chromosome 32. CONCLUSION: SIDER2 elements constitute a relevant piece in the Leishmania genome organization. Sequence characteristics, genomic distribution and evolutionarily conservation of SIDER2s are suggestive of relevant functions for these elements in Leishmania. Apart from a proved involvement in post-transcriptional mechanisms of gene regulation, SIDER2 elements could be involved in DNA amplification processes and, perhaps, in chromosome segregation as centromeric sequences.

The L1Tc C-terminal domain from Trypanosoma cruzi non-long terminal repeat retrotransposon codes for a protein that bears two C2H2 zinc finger motifs and is endowed with nucleic acid chaperone activity.

L1Tc, a non-long terminal repeat retrotransposon from Trypanosoma cruzi, is a 4.9-kb actively transcribed element which contains a single open reading frame coding for the machinery necessary for its autonomous retrotransposition. In this paper, we analyze the protein encoded by the L1Tc 3' region, termed C2-L1Tc, which contains two zinc finger motifs similar to those present in the TFIIIA transcription factor family. C2-L1Tc binds nucleic acids with different affinities, such that RNA > tRNA > single-stranded DNA > double-stranded DNA, without any evidence for sequence specificity. C2-L1Tc also exhibits nucleic acid chaperone activity on different DNA templates that may participate in the mechanism of retrotransposition of the element. C2-L1Tc promotes annealing of complementary oligonucleotides, prevents melting of perfect DNA duplexes, and facilitates the strand exchange between DNAs to form the most stable duplex DNA in competitive displacement assays. Mapping of regions of C2-L1Tc using specific peptides showed that nucleic acid chaperone activity required a short basic sequence accompanied by a zinc finger motif or by another basic region such as RRR. Thus, a short basic polypeptide containing the two C(2)H(2) motifs promotes formation of the most stable duplex DNA at a concentration only three times higher than that required for C2-L1Tc.

Transcriptional Profiling of Immune-Related Genes in Leishmania infantum-Infected Mice: Identification of Potential Biomarkers of Infection and Progression of Disease.

Leishmania spp. is a protozoan parasite that affects millions of people around the world. At present, there is no effective vaccine to prevent leishmaniases in humans. A major limitation in vaccine development is the lack of precise understanding of the particular immunological mechanisms that allow parasite survival in the host. The parasite-host cell interaction induces dramatic changes in transcriptome patterns in both organisms, therefore, a detailed analysis of gene expression in infected tissues will contribute to the evaluation of drug and vaccine candidates, the identification of potential biomarkers, and the understanding of the immunological pathways that lead to protection or progression of disease. In this large-scale analysis, differential expression of 112 immune-related genes has been analyzed using high-throughput qPCR in spleens of infected and naïve Balb/c mice at four different time points. This analysis revealed that early response against Leishmania infection is characterized by the upregulation of Th1 markers and M1-macrophage activation molecules such as Ifng, Stat1, Cxcl9, Cxcl10, Ccr5, Cxcr3, Xcl1, and Ccl3. This activation doesn't protect spleen from infection, since parasitic burden rises along time. This marked difference in gene expression between infected and control mice disappears during intermediate stages of infection, probably related to the strong anti-inflammatory and immunosuppresory signals that are activated early upon infection (Ctla4) or remain activated throughout the experiment (Il18bp). The overexpression of these Th1/M1 markers is restored later in the chronic phase (8 wpi), suggesting the generation of a classical "protective response" against leishmaniasis. Nonetheless, the parasitic burden rockets at this timepoint. This apparent contradiction can be explained by the generation of a regulatory immune response characterized by overexpression of Ifng, Tnfa, Il10, and downregulation Il4 that counteracts the Th1/M1 response. This large pool of data was also used to identify potential biomarkers of infection and parasitic burden in spleen, on the bases of two different regression models. Given the results, gene expression signature analysis appears as a useful tool to identify mechanisms involved in disease outcome and to establish a rational approach for the identification of potential biomarkers useful for monitoring disease progression, new therapies or vaccine development.