Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins

Background:Trypanosoma cruzi ribosomal P proteins, P2β and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC). It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with β1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals.Methodology/Principal findings:We evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC) stimulated with P2β, the C-terminal portion of P0 (CP0) proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2β or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-α and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells.Conclusions/Significance:Our results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-α and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T. cruzi infection.Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Atienza, Augusto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Perez Prados, Graciela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: Buying, Alcinette. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Santos, Radleigh. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Tasso, Laura Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bonato, Ricardo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Chiale, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Pinilla, Clemencia. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Judkowski, Valeria A.. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Silencing cytokeratin 18 gene inhibits intracellular replication of Trypanosoma cruzi in HeLa cells but not binding and invasion of trypanosomes

<p>Abstract</p> <p>Background</p> <p>As an obligatory intracellular parasite, <it>Trypanosoma cruzi</it>, the etiological agent of Chagas' disease, must invade and multiply within mammalian cells. Cytokeratin 18 (CK18) is among the host molecules that have been suggested as a mediator of important events during <it>T. cruzi</it>-host cell interaction. Based on that possibility, we addressed whether RNA interference (RNAi)-mediated down regulation of the CK18 gene could interfere with the parasite life cycle <it>in vitro</it>. HeLa cells transiently transfected with CK18-RNAi had negligible levels of CK18 transcripts, and significantly reduced levels of CK18 protein expression as determined by immunoblotting or immunofluorescence.</p> <p>Results</p> <p>CK18 negative or positive HeLa cells were invaded equally as well by trypomastigotes of different <it>T. cruzi </it>strains. Also, in CK18 negative or positive cells, parasites recruited host cells lysosomes and escaped from the parasitophorous vacuole equally as well. After that, the growth of amastigotes of the Y or CL-Brener strains, was drastically arrested in CK18 RNAi-treated cells. After 48 hours, the number of amastigotes was several times lower in CK18 RNAi-treated cells when compared to control cells. Simultaneous staining of parasites and CK18 showed that in HeLa cells infected with the Y strain both co-localize. Although the amastigote surface protein-2 contains the domain VTVXNVFLYNR previously described to bind to CK18, in several attempts, we failed to detect binding of a recombinant protein to CK-18.</p> <p>Conclusion</p> <p>The study demonstrates that silencing CK18 by transient RNAi, inhibits intracellular multiplication of the Y and CL strain of <it>T. cruzi </it>in HeLa cells, but not trypanosome binding and invasion.</p

Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy

Subdominant/Cryptic CD8 T Cell Epitopes Contribute to Resistance against Experimental Infection with a Human Protozoan Parasite

During adaptive immune response, pathogen-specific CD8+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8+ T cells of H-2a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8+ T-Cell Response: Reversal by Adenoviral Vaccine

MHC class Ia-restricted CD8+ T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8+ T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8+ T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8+ T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8+ T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8+ cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8+ T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination

Canagliflozin and Renal Outcomes in Type 2 Diabetes and Nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to 300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m 2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Ageing and Financial Stability

Abstract: Although the precise details are subject to major uncertainty, it seems likely that the process of population ageing will involve major shifts in financing, which may give rise to financial turbulence and systemic risk. The locus and scale of these effects will also depend on the predominant approach to retirement income provision. It is argued that the financial-stability risks arising from continuing with unsustainable pay-as-you-go systems would be more threatening than those arising from funding. Fiscal crises can have incalculable consequences for private financial markets, while pension funding involves more an adaptation by regulatory authorities to a more securitised and institutionalised financial system, that is likely to develop in any case. Concerning policy, for social security, the key issue is reform, so that the fiscal difficulties and their consequences for financial stability foreshadowed above do not arise. For institutional investors involved in funding, policy issues arising include the need for prudent person asset regulation, absence of guarantees generating moral hazard and international diversification of institutional portfolios, so that they are less dependent on the performance of the domestic economy than would otherwise be the case. Banks would not be immune to the side-effects of the various patterns ageing will generate, and an awareness of such risks as well a