Chagas disease, caused by the protozoan Trypanosoma cruzi , remains a major public health challenge affecting millions in Latin America and worldwide. Although significant progress has been made in ...vector control, no vaccine exists to prevent infection or mitigate disease pathogenesis. We developed a rationally designed chimeric protein vaccine, N-Tc52/TSkb20, incorporating immunodominant epitopes from two T. cruzi antigens, the amino-terminal portion of Tc52 and the TSkb20 epitope derived from trans-sialidase. The objectives of this study were to construct and characterize the antigen and evaluate its protective potential in an immunoprophylactic murine model of T. cruzi infection. The N-Tc52/TSkb20 protein was recombinantly expressed in E. coli and its identity was confirmed using mass spectrometry and Western blotting. Immunization with the chimeric protein significantly controlled parasitemia and reduced the heart, colon, and skeletal muscle parasite burdens compared to non-vaccinated mice. Protection was superior to vaccination with the individual parental antigen components. Mechanistically, the vaccine induced potent CD8+ T-cell and IFNγ responses against the incorporated epitopes and a protective IgG antibody profile. A relatively low IL-10 response favored early parasite control. These results validate the promising multi-epitope approach and support the continued development of this type of rational vaccine design strategy against Chagas disease.
Previous studies showed that a naturally attenuated strain from Trypanosoma cruzi triggers an immune response mainly related to a Th2-type profile. Albeit this, a strong protection against virulent ...challenge was obtained after priming mice with this attenuated strain. However, this protection is not enough to completely clear parasites from the host. In T. cruzi infection, early Interferon-gamma (IFN-γ) is critical to lead type 1 responses able to control intracellular parasites. Therefore we evaluated whether the co-administration of a plasmid encoding murine IFN-γ could modify the immune response induced by infection with attenuated parasites and improve protection against further infections.
C57BL/6J mice were infected intraperitoneally with three doses of live attenuated parasites in combination with plasmid pVXVR-mIFN-γ. Before each infection dose, sera samples were collected for parasite specific antibodies determination and cytokine quantification. To evaluate the recall response to T. cruzi, mice were challenged with virulent parasites 30 days after the last dose and parasite load in peripheral blood and heart was evaluated.
As determined by ELISA, significantly increase in T. cruzi specific antibodies response was detected in the group in which pVXVR-mIFN-γ was incorporated, with a higher predominance of IgG2a subtype in comparison to the group of mice only inoculated with attenuated parasites. At our limit of detection, serum levels of IFN-γ were not detected, however a slight decrease in IL-10 concentrations was observed in groups in which pVXVR-mIFN-γ was supplemented. To analyze if the administration of pVXVR-mIFN-γ has any beneficial effect in protection against subsequent infections, all experimental groups were submitted to a lethal challenge with virulent bloodstream trypomastigotes. Similar levels of challenge parasites were detected in peripheral blood and heart of mice primed with attenuated parasites alone or combined with plasmid DNA. Expansion of IgG antibodies was not significant in TCC+ pVXVR-mIFN-γ; however, the overall tendency to sustain a Th2 profile was maintained.
Overall, these results suggest that administration of plasmid pVXVR-mIFN-γ could have beneficial effects on host specific antibody production in response to T. cruzi attenuated infection; however, this outcome is not reflected in an improved protection against further virulent infections.
To date, there is no specific literature available on the determinants for therapeutic failure (TF) with meglumine antimoniate (MA) in Northwestern-Argentina. This study aimed to identify ...epidemiological, clinical, and treatment-related factors that could be involved in TF.
We performed a case-control study. Cases were represented by patients who showed TF after administration of the first course of MA treatment, whereas, controls were determined as patients who evolved towards healing after the first MA cycle received. Crude Odds Ratios and their corresponding 90% confidence intervals (CI) were calculated, and risk factors were then tested by multivariate analysis using logistic binary regression. Three hundred and eighty-four patients with a presumptive diagnosis of ACL were recruited, and 153 with a positive diagnosis were selected. We included in the study 71 patients, who underwent specific treatment with MA, presented complete data on response to treatment, and had a minimum post-treatment follow-up of 6 months in cutaneous leishmaniasis, and 12 months in mucosal leishmaniasis. Of these, 34 (47.9%) presented TF. In the initial analysis, TF was significantly associated with the geographical area of disease acquisition (p = 0.036), the presence of mucosal lesions (p = 0.042), the presence of concomitant skin and mucosal lesions (p = 0.002), and lesion age ≥ 6 months (p = 0.018). Risk factors influencing TF in the final multivariate model included the geographical area where the disease was acquired (adjusted Odd Ratio 8.062; 95% CI 1.914-33.959; p = 0.004), and lesion age ≥ 6 months (adjusted Odd Ratio 10.037; 95% CI 1.383-72.843; p = 0.023).
The results of the present study suggest the existence of some risk factors linked to TF in Northwestern-Argentina, which deserve further investigation. Herein we recorded a high percentage of TF and we described clinical and epidemiological characteristics associated with TF that could be taken into account improving the clinical management of patients.
Antimonials continue to be considered the first-line treatment for leishmaniases, but its use entails a wide range of side effects and serious reactions. The search of new drugs requires the ...development of methods more sensitive and faster than the conventional ones. We developed and validated a fluorescence assay based in the expression of tdTomato protein by
, and we applied this method to evaluate the activity
of flavonoids and reference drugs. The pIR1SAT/tdTomato was constructed and integrated into the genome of
Parasites were selected with nourseothricin (NTC). The relation of
/tc3 fluorescence and the number of parasites was determined; then the growth
and infectivity in BALB/c mice was characterized. To validate the fluorescence assay, the efficacy of miltefosine and meglumine antimoniate was compared with the conventional methods. After that, the method was used to assess
the activity of flavonoids; and the mechanism of action of the most active compound was evaluated by transmission electron microscopy and ELISA. A linear correlation was observed between the emission of fluorescence of
/tc3 and the number of parasites (r
= 0.98), and the fluorescence was stable in the absence of NTC. No differences were observed in terms of infectivity between
/tc3 and wild strain. The efficacy of miltefosine and meglumine antimoniate determined by the fluorescence assay and the microscopic test showed no differences, however,
the fluorescence assay was more sensitive than limiting dilution assay. Screening assay revealed that the flavonoid galangin (GAL) was the most active compound with IC
values of 53.09 µM and 20.59 µM in promastigotes and intracellular amastigotes, respectively. Furthermore, GAL induced mitochondrial swelling, lipid inclusion bodies and vacuolization in promastigotes; and up-modulated the production of IL-12 p70 in infected macrophages. The fluorescence assay is a useful tool to assess the anti-leishmanial activity of new compounds. However, the assay has some limitations in the macrophage-amastigote model that might be related with an interfere of flavanol aglycones with the fluorescence readout of tdTomato. Finally, GAL is a promising candidate for the development of new treatment against the leishmaniasis.
Subjects are considered infected with
when tested positive by at least two out of three serological tests, whereas a positive result in only one of up to three tests is termed "serodiscordant" (SD). ...Assessment of parasite-specific T-cell responses may help discriminate the uninfected from infected individuals among SD subjects.
Peripheral blood mononuclear cells from SD and seropositive (SP) subjects, who were born in areas endemic for
infection but living in Buenos Aires city, Argentina, at the time of the study, and seronegative unexposed subjects were included for analysis. The function and phenotype of T cells were assessed by interferon-γ (IFN-γ) and interleukin (IL)-2 enzyme-linked immunospot assay and multiparameter flow cytometry.
-specific antibodies were quantified by conventional serology and a multiplex assay format.
SD subjects exhibited immunity cell responses to
but in contrast to SP subjects, T cells in SD subjects more often display the simultaneous production of IFN-γ and IL-2 in response to
antigens and have a resting phenotype. SD individuals also have higher IFN-γ spot counts, polyfunctional CD4
T-cells enriched in IL-2 secreting cells and low levels of antibodies specific for a set of
-derived recombinant proteins compared with the SP group. Long-term follow-up of SD individuals confirmed that humoral and T-cell responses fluctuate but are sustained over time in these subjects. T cells in SD subjects for
infection did not recognize
antigens.
Both T-cell and humoral responses in most subjects assessed by conventional tests as SD for
infection indicate prior exposure to infection and the establishment of immunological memory suggestive of a resolved infection.
•HCV-RNA is consistently recovered during PBMC culture from HCV infected patients.•In this study, the evolution of HCV during PBMC culture over time was showed.•This work evidence PBMC as an ...extrahepatic compartment where replication occurs.•PBMC could produce HCV distinctive variants.•The HCV variants in PBMC could have implications in therapy and liver disease.
Even though hepatocytes are the main site for hepatitis C virus (HCV) replication, peripheral blood mononuclear cells (PBMC) have also been proposed as a suitable site for HCV replication. However, this issue still remains under discussion. We have previously developed an innovative system where HCV-RNA can be recovered during PBMC culture from HCV infected patients. Thus, the aim of this work was to use this novel approach in order to observe the evolution and replication of HCV genotype 1b in the PBMC of an HIV-HCV coinfected patient.
HCV-RNA was extracted from serum, uncultured PBMC and PBMC culture at day 6, 20 and 33. The evolutionary analysis was performed using the direct sequences of three viral regions: 5′UTR, E2 and NS5A. Additionally, E2 region was cloned in order to extend the evolutive analysis.
In the present work, the molecular characterization of HCV along the culture showed a clear dynamic evolving process with the appearance of several nucleotide or amino acid changes in the three regions analyzed. Furthermore, the population analysis of E2 clones showed emerging and loss of lineages which indicate the fast evolutive dynamics of this system.
Since evolution can take place only if the virus is replicating in the culture, this finding constitutes an important evidence of viral replication in PBMC. Moreover, this extrahepatic compartment could be very important due to the presence of distinctive variants that could be responsible for resistance to treatment, viral pathogenesis and other clinical implications.
Resilient overlay design in DWDM systems Parodi, Cecilia; Robledo, Franco; Romero, Pablo ...
Yugoslav Journal of Operations Research,
2016, Volume:
26, Issue:
3
Journal Article
Peer reviewed
Open access
The goal of this work is to design a minimum cost resilient overlay network,
where a data network is on top of a transport network. Two major challenges
are addressed. On one hand, a single failure ...in the transport network causes
multiple simultaneous failures; on the other, the multicommodity flow must
respect integrality. An integer programming formulation is presented to
design an overlay, meeting the previous constraints. We prove the problem
belongs to the class NP-Hard. Then, a decomposition approach is introduced,
where the problem is solved in two steps by means of relaxations of the
original formulation. Experiments carried out with real-life instances,
coming from the Uruguayan telecommunication operator, show that the approach
is competitive with respect to previous metaheuristics, to know, Tabu-Search
(TS) and Variable Neighborhood Search (VNS). A modest percentage of
cost-reduction is achieved in some instances, which means millionaire savings
in practice.
nema
Protective Immunity Against Trypanosoma Cruzi Parodi, Cecilia; Padilla, Angel Marcelo; Basombrío, Miguel Angel
Memórias do Instituto Oswaldo Cruz,
07/2009, Volume:
104, Issue:
s1
Journal Article
Peer reviewed
Open access
Upon infection, Trypanosoma cruzi triggers a strong immune response
that has both protective and pathological consequences. In this work,
several important questions regarding protective immunity are ...reviewed.
Emphasis is placed on recent studies of the important protective role
of CD8+ T cells and on previous studies of immunisation of domestic T.
cruzi reservoirs that sought to address practical vaccination problems.
Research on the maturation of memory cells and studies indicating that
the prevalence of T. cruzi-specific T-cell responses and a high
frequency of committed CD8+ T cells are associated with better clinical
outcomes are also reviewed. Additionally, animal models in which
protection was achieved without immunopathological consequences are
discussed.
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