Priscila C. Teixeira

Immunological and non-immunological effects of cytokines and chemokines in the pathogenesis of chronic Chagas disease cardiomyopathy

The pathogenesis of Chagas disease cardiomyopathy (CCC) is not well understood. Since studies show that myocarditis is more frequent during the advanced stages of the disease, and the prognosis of CCC is worse than that of other dilated cardiomyopathies of non-inflammatory aetiology, which suggest that the inflammatory infiltrate plays a major role in myocardial damage. In the last decade, increasing evidence has shown that inflammatory cytokines and chemokines play a role in the generation of the inflammatory infiltrate and tissue damage. CCC patients have an increased peripheral production of the inflammatory Th1 cytokines IFN-gamma and TNF-alpha when compared to patients with the asymptomatic/indeterminate form. Moreover, Th1-T cells are the main producers of IFN-gamma and TNF-alpha and are frequently found in CCC myocardial inflammatory infiltrate. Over the past several years, our group has collected evidence that shows several cytokines and chemokines produced in the CCC myocardium may also have a non-immunological pathogenic effect via modulation of gene and protein expression in cardiomyocytes and other myocardial cell types. Furthermore, genetic polymorphisms of cytokine, chemokine and innate immune response genes have been associated with disease progression. We will review the molecular and immunological mechanisms of myocardial damage in human CCC in light of recent findings.

Heterozygosity for the S180L Variant of MAL/TIRAP, a Gene Expressing an Adaptor Protein in the Toll‐Like Receptor Pathway, Is Associated with Lower Risk of Developing Chronic Chagas Cardiomyopathy

BACKGROUND Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. Among T. cruzi-infected individuals, only a subgroup develops severe chronic Chagas cardiomyopathy (CCC); the majority remain asymptomatic. T. cruzi displays numerous ligands for the Toll-like receptors (TLRs), which are an important component of innate immunity that lead to the transcription of proinflammatory cytokines by nuclear factor-kappaB. Because proinflammatory cytokines play an important role in CCC, we hypothesized that single-nucleotide polymorphisms (SNPs) in the genes that encode proteins in the TLR pathway could explain differential susceptibility to CCC among T. cruzi-infected individuals. METHODS For 169 patients with CCC and 76 T. cruzi-infected, asymptomatic individuals, we analyzed SNPs by use of polymerase chain reaction-restriction fragment length polymorphism analysis for the genes TLR1, TLR2, TLR4, TLR5, TLR9, and MAL/TIRAP, which encodes an adaptor protein. RESULTS Heterozygous carriers of the MAL/TIRAP variant S180L were more prevalent in the asymptomatic group (24 [32%] of 76 subjects) than in the CCC group (21 [12%] of 169) (chi2=12.6; P=.0004 [adjusted P (Pc)=.0084]; odds ratio [OR], 0.31 [95% confidence interval {CI}, 0.16-0.60]). Subgroup analysis showed a stronger association when asymptomatic patients were compared with patients who had severe CCC (i.e., patients with left-ventricular ejection fraction<or=40%) (chi2=11.3; P=.0008 [Pc=.017]; OR, 0.22 [95% CI, 0.09-0.56]) than when asymptomatic patients were compared with patients who had mild CCC (i.e., patients with left-ventricular ejection fraction>40%) (chi2=7.7; P=.005 [Pc=.11]; OR, 0.33 [95% CI, 0.15-0.73]). CONCLUSION T. cruzi-infected individuals who are heterozygous for the MAL/TIRAP S180L variant that leads to a decrease in signal transduction upon ligation of TLR2 or TLR4 to their respective ligand may have a lower risk of developing CCC.

DNA damage by 3,6-dihydropyrazine-2,5-dipropanoic acid, the cyclic dimerization product of 5-aminolevulinic acid.

Abstract 5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in lead poisoning and inborn porphyrias. It has been shown to produce reactive oxygen species upon metalcatalyzed aerobic oxidation and to cause oxidative damage to proteins, liposomes, DNA, and subcellular structures. Studies have also shown that ALA may condense to yield the cyclic product 3,6-dihydropyrazine 2,5-dipropanoic acid (DHPY). Here we propose that DHPY could be involved in DNA damage in the presence of high concentrations of ALA. Exposure of plasmid pUC19 DNA to low concentrations of DHPY (2 10 M) in the presence of 0.1 mM Cu[2+] ions causes DNA strand breaks, as demonstrated by agarose gel electrophoresis. It was also shown that in the presence of Cu[2+] ions DHPY is able to increase the oxidation of monomeric 2deoxyguanosine to form 8- oxo-7,8-dihydro-2deoxyguanosine as inferred from high performance liquid chromatography measurements using electrochemical detection. Addition of a metal chelator (bathocuproine, 0.5 mM), the DNA compacting polyamines spermidine (1 mM) and spermine (1 mM) or antioxidant enzymes such as superoxide dismutase (10 g/ml) and catalase (20 g/ml) protect the DNA against these damages. The data presented here are discussed with respect to the increased frequency of liver cancer in patients with acute intermittent porphyria.

Polymorphism in the Alpha Cardiac Muscle Actin 1 Gene Is Associated to Susceptibility to Chronic Inflammatory Cardiomyopathy

Aims Chagas disease, caused by the protozoan Trypanosoma cruzi is endemic in Latin America, and may lead to a life-threatening inflammatory dilated, chronic Chagas cardiomyopathy (CCC). One third of T. cruzi-infected individuals progress to CCC while the others remain asymptomatic (ASY). A possible genetic component to disease progression was suggested by familial aggregation of cases and the association of markers of innate and adaptive immunity genes with CCC development. Since mutations in multiple sarcomeric genes, including alpha-cardiac actin (ACTC1) have been involved in hereditary dilated cardiomyopathy, we investigated the involvement of the ACTC1 gene in CCC pathogenesis. Methods and Results We conducted a proteomic and genetic study on a Brazilian study population. The genetic study was done on a main cohort including 118 seropositive asymptomatic subjects and 315 cases and the replication was done on 36 asymptomatic and 102 CCC cases. ACTC1 protein and mRNA levels were lower in myocardial tissue from patients with end-stage CCC than those found in hearts from organ donors. Genotyping a case-control cohort of CCC and ASY subjects for all informative single nucleotide polymorphism (SNP) in the ACTC1 gene identified rs640249 SNP, located at the 5’ region, as associated to CCC. Associations are borderline after correction for multiple testing. Correlation and haplotype analysis led to the identification of a susceptibility haplotype. Functional assays have shown that the rs640249A/C polymorphism affects the binding of transcriptional factors in the promoter regions of the ACTC1 gene. Confirmation of the detected association on a larger independent replication cohort will be useful. Conclusions Genetic variations at the ACTC1 gene may contribute to progression to chronic Chagas Cardiomyopathy among T. cruzi-infected patients, possibly by modulating transcription factor binding to ACTC1 promoter regions.

Danos ao DNA promovidos por ácido 5-aminolevulínico: possível associação com o desenvolvimento de carcinoma hepatocelular em portadores de porfiria aguda intermitente

5-Aminolevulinic acid (ALA) is a heme precursor accumulated in acute intermittent porphyria (AIP), which might be associated with hepatocellular carcinoma (HCC) in symptomatic patients. Under metal catalyzed oxidation, ALA and its cyclic dimerization product, 3,6-dihydropyrazine-2,5-dipropanoic acid, produce reactive oxygen species that damage plasmid and calf thymus DNA bases, increase the steady state level of 8-oxo-7,8-dihydro-2´-deoxyguanosine in liver DNA and promote mitochondrial DNA damage. The final product of ALA, 4,5-dioxovaleric acid (DOVA), is able to alkylate guanine moieties, producing adducts. ALA and DOVA are mutagenic in bacteria. This review shows an up-to-date literature data that reinforce the hypothesis that the DNA damage induced by ALA may be associated with the development of HCC in AIP patients.

Proteins differentially expressed in human beta-cells-enriched pancreatic islet cultures and human insulinomas ☆

In view of the great demand for human beta-cells for physiological and medical studies, we generated cell lines derived from human insulinomas which secrete insulin, C-peptide and express neuroendocrine and islet markers. In this study, we set out to characterize their proteomes, comparing them to those of primary beta-cells using DIGE followed by MS. The results were validated by Western blotting. An average of 1800 spots was detected with less than 1% exhibiting differential abundance. Proteins more abundant in human islets, such as Caldesmon, are involved in the regulation of cell contractility, adhesion dependent signaling, and cytoskeletal organization. In contrast, almost all proteins more abundant in insulinoma cells, such as MAGE2, were first described here and could be related to cell survival and resistance to chemotherapy. Our proteomic data provides, for the first time, a molecular snapshot of the orchestrated changes in expression of proteins involved in key processes which could be correlated with the altered phenotype of human beta-cells. Collectively our observations prompt research towards the establishment of bioengineered human beta-cells providing a new and needed source of cultured human beta-cells for beta-cell research, along with the development of new therapeutic strategies for detection, characterization and treatment of insulinomas.