Rubén Laguens

Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation.

BACKGROUNDnOne hundred years after the discovery of Chagas disease, it remains a major neglected tropical disease. Chronic Chagas heart disease (cChHD) is the most severe manifestation. Heart transplantation is the proper treatment for end-stage heart failure, although reactivation of disease may result after receipt of immunosuppressive therapy. T. cruzi strains cluster into 6 discrete typing units (DTUs; I-VI) associated with different geographical distribution, transmission cycles and varying disease symptoms. In the southern cone of South America, T. cruzi II, V, and VI populations appear to be associated with Chagas disease and T. cruzi I with sylvatic cycles.nnnMETHODSnMolecular characterization of DTUs, T. cruzi I genotypes (on the basis of spliced-leader gene polymorphisms), and minicircle signatures was conducted using cardiac explant specimens and blood samples obtained from a cohort of 16 Argentinean patients with cChHD who underwent heart transplantation and from lesion samples obtained from 6 of these patients who presented with clinical reactivation of Chagas disease.nnnRESULTSnParasite persistence was associated with myocarditis progression, revealing T. cruzi I (genotype Id) in 3 explant samples and T. cruzi II, V, or VI in 5 explant samples. Post-heart transplantation follow-up examination of bloodstream DTUs identified T. cruzi I in 5 patients (genotypes Ia or Id) and T. cruzi II, V, or VI in 7 patients. T. cruzi I, V, and VI were detected in skin chagoma specimens, and T. cruzi V and VI were detected in samples obtained from patients with myocarditis reactivations. Multiple DTUs or genotypes at diverse body sites and polymorphic minicircle signatures at different cardiac regions revealed parasite histotropism. T. cruzi I infections clustered in northern Argentina (latitude, 23 degrees S-27 degrees S), whereas T. cruzi II, V, or VI DTUs were more ubiquitous.nnnCONCLUSIONSnMultiple DTUs coexist in patients with Chagas disease. The frequent finding of T. cruzi I associated with cardiac damage was astounding, revealing its pathogenic role in cChHD at the southern cone.

Early diagnosis of recurrence of Trypanosoma cruzi infection by polymerase chain reaction after heart transplantation of a chronic Chagas' heart disease patient.

Heart transplantation is contraindicated as an effective treatment for end-stage Chagas heart disease because of post-operative recurrence of Trypanosoma cruzi infection and reactivation of disease after immunosupression. In a follow-up study of a heart transplanted patient with Chagas disease, we prospectively evaluated the usefulness of the polymerase chain reaction (PCR) for early diagnosis of reactivation. We monitored post-operative recurrence of Trypanosoma cruzi infection with microscopic observation of the parasite in peripheral blood (Strouts method), endomyocardial biopsies (EMBs), skin lesions, and 2 PCR assays, based on the amplification of specific T cruzi kinetoplastid and nuclear DNA sequences. During follow-up, parasite DNA was amplified in blood samples and EMB sections 41 days before we observed patent parasitemia and cutaneous manifestations of reactivation, proving that PCR is much more sensitive than direct microscopic observation for early diagnosis of disease reactivation in heart-transplanted Chagas disease patients.

Plasmid-mediated VEGF gene transfer induces cardiomyogenesis and reduces myocardial infarct size in sheep

We have recently reported that in pigs with chronic myocardial ischemia heart transfection with a plasmid encoding the 165 isoform of human vascular endothelial growth factor (pVEGF165) induces an increase in the mitotic index of adult cardiomyocytes and cardiomyocyte hyperplasia. On these bases we hypothesized that VEGF gene transfer could also modify the evolution of experimental myocardial infarct. In adult sheep pVEGF165 (3.8u2009mg, n=7) or empty plasmid (n=7) was injected intramyocardially 1u2009h after coronary artery ligation. After 15 days infarct area was 11.3±1.3% of the left ventricle in the VEGF group and 18.2±2.1% in the empty plasmid group (P<0.02). The mechanisms involved in infarct size reduction (assessed in additional sheep at 7 and 10 days after infarction) included an increase in early angiogenesis and arteriogenesis, a decrease in peri-infarct fibrosis, a decrease in myofibroblast proliferation, enhanced cardiomyoblast proliferation and mitosis of adult cardiomyocytes with occasional cytokinesis. Resting myocardial perfusion (99mTc-sestamibi SPECT) was higher in VEGF-treated group than in empty plasmid group 15 days after myocardial infarction. We conclude that plasmid-mediated VEGF gene transfer reduces myocardial infarct size by a combination of effects including neovascular proliferation, modification of fibrosis and cardiomyocyte regeneration.

Trypanosoma cruzi: identification of a galactose-binding protein that binds to cell surface of human erythrocytes and is involved in cell invasion by the parasite

Trypanosoma cruzi must invade mammalian host cells to replicate and complete its life cycle. Almost all nucleated mammalian cells can be invaded by the parasite following a receptor-ligand recognition as an early prerequisite. In this work, we describe a 67-kDa lectin-like glycoprotein that binds to desialylated human erythrocyte membranes in a galactose-dependent way. This protein is present on the parasite surface in both infective and non-infective stages of T. cruzi. More interestingly, we demonstrate by lectin-immuno-histochemistry assays that the 67kDa protein is involved in the recognition of host-cell receptors in mouse cardiac tissue and human cardiac aortic endothelium and mammary artery tissue. Moreover, antibodies against the 67kDa glycoprotein inhibit in vitro host-cell invasion by 63%. These data suggest that the 67kDa glycoprotein in vivo is needed for host-cell invasion by T. cruzi.

Repeated, but not single, VEGF gene transfer affords protection against ischemic muscle lesions in rabbits with hindlimb ischemia.

Vascular endothelial growth factor (VEGF) gene transfer-mediated angiogenesis has been proposed for peripheral artery disease. However, protocols using single administration have shown little benefit. Given that the transient nature of VEGF gene expression provokes instability of neovasculature, we hypothesized that repeated administration would provide efficient tissue protection. We thus compared single vs repeated transfection in a rabbit model of hindlimb ischemia by injecting a plasmid encoding human VEGF165 (pVEGF165) at 7 (GI, n=10) or 7 and 21 (GII, n=10) days after surgery. Placebo animals (GIII, n=10) received empty plasmid. Fifty days after surgery, single and repeated administration similarly increased saphenous peak flow velocity and quantity of angiographically visible collaterals. However, microvasculature increased only with repeated transfection: capillary density was 49.4±15.4 capillaries per 100 myocytes in GI, 84.6±14.7 in GII (P<0.01 vs GI and GIII) and 49.3±13.6 in GIII, and arteriolar density was 1.9±0.6 arterioles per mm2 in GI, 3.0±0.9 in GII (P<0.01 vs GI and GIII) and 1.5±0.6 in GIII. Muscle lesions were reduced only within repeated transfection. With single administration, gene expression peaked at 7 days and declined rapidly, but with repeated administration, it remained positive at 50 days. At 90 days of repeated transfection (additional animals), gene expression decreased significantly, but neovessel densities did not. Thus, repeated, but not single, VEGF gene transfection resulted in increased microvasculature, which, in turn, afforded effective protection against ischemic muscle damage.

Production of functional platelet-like particles by the megakaryoblastic DAMI cell line provides a model for platelet biogenesis

The aim of this study was to evaluate cell maturation and the platelet production capacity of the megakaryoblastic DAMI cell line, to characterize platelet-like particles produced and to investigate the mechanisms involved in their production. DAMI cell maturation was induced by phorbol myristate acetate (PMA) and thrombopoietin (TPO). Expression levels of GATA-1, Fli-1 and NF-E2 were evaluated using real-time PCR and western blot. Platelet-like particles were characterized by the presence of GPIb and GPIIb by flow cytometry, while the soluble fragment of GPIb, glycocalicin, was detected by enzyme immunoassay. Dense and alpha granules were evaluated by mepacrine staining and thrombospondin-1 detection, respectively, and by electron microscopy. Functional capacity of platelet-like particles was studied by measuring P-selectin membrane after thrombin stimulation by flow cytometry and actin polymerization using phalloidin-FITC by immunofluorescence. We found that stimulation of DAMI cells with high concentration of PMA and TPO induced the expression of transcription factors GATA-1 and Fli-1 followed by an increase in the isoform a of NF-E2. Mature DAMI cells give rise to extensions resembling proplatelets and later, produce platelet-like particles expressing GPIIb and GPIb on their surface and containing dense and alpha granules, which were confirmed by electron microscopy. Platelet functionality was demonstrated by the increase in P-selectin membrane expression after thrombin stimulation and by their ability to spread on fibrinogen matrices. DAMI cell line induced to differentiate into mature megakaryocytes is able to produce functional platelets providing a suitable model to study the mechanisms involved in platelet generation.

Presence of vulnerable coronary plaques in middle-aged individuals who suffered a brain death

AIMSnVulnerable plaques in coronary arteries are frequently found in individuals who died suddenly or due to an acute coronary syndrome. The prevalence and characteristics of these plaques in the middle-aged apparently healthy population are unknown.nnnMETHODS AND RESULTSnFrom a total of 652 hearts from transplant donors collected between 1996 and 2007, we selected those from apparently healthy individuals older than 40 years old who died of head trauma or stroke and had no evidence of prior vascular diseases. The coronary arteries were examined by serial sectioning at 3 mm intervals, and all areas of cross-sectional luminal narrowing were processed for histological, immunohistochemical, and morphometric studies. The atherosclerotic plaques were classified according to the American Heart Association Report. A total of 160 hearts were examined. Mean age was 50.3 +/- 5.8 years. Sixty-eight hearts had no advanced coronary atherosclerotic lesions (Type I, II, and III of the American Heart classification). In the remaining 92 hearts, we found 179 plaques considered high-risk lesions (American Heart Association Type IV, V, and VI). These plaques were more frequently found in males (P < 0.001) and in those with a higher heart weight (P < 0.001). The median (25th and 75th percentiles) vascular narrowing value using a planimetric analysis was 32% (21-53). No significant association with the cause of death was found (P = 0.09).nnnCONCLUSIONnHigh-risk coronary artery plaques not associated with significant vascular lumen reduction exist in 57% of patients who suffered a brain death with a mean of 1.11 lesions prone to rupture per individual.

Presence of antigen-experienced T cells with low grade of differentiation and proliferative potential in chronic Chagas disease myocarditis

Background The main consequence of chronic Trypanosoma cruzi infection is the development of myocarditis in approximately 20–30% of infected individuals but not until 10–20 years after the initial infection. We have previously shown that circulating interferon-γ-secreting T cells responsive to Trypanosoma cruzi antigens in chronic Chagas disease patients display a low grade of differentiation and the frequency of these T lymphocytes decreases along with the severity of heart disease. This study thought to explore the expression of inhibitory receptors, transcription factors of type 1 or regulatory T cells, and markers of T cell differentiation, immunosenescence or active cell cycle in cardiac explants from patients with advanced Chagas disease myocarditis. Methodology/Principal Findings The expression of different markers for T and B cells as well as for macrophages was evaluated by immunohistochemistry and immunofluorescence techniques in cardiac explants from patients with advanced chronic Chagas disease submitted to heart transplantation. Most infiltrating cells displayed markers of antigen-experienced T cells (CD3+, CD4+, CD8+, CD45RO+) with a low grade of differentiation (CD27+, CD57−, CD45RA−, PD-1−). A skewed T helper1/T cytotoxic 1 profile was supported by the expression of T-bet; whereas FOXP3+ cells were scarce and located only in areas of severe myocarditis. In addition, a significant proliferative capacity of CD3+ T cells, assessed by Ki67 staining, was found. Conclusions/Significance The quality of T cell responses and immunoregulatory mechanisms might determine the pattern of the cellular response and the severity of disease in chronic Trypanosoma cruzi infection.

Differences in resistance to reinfection with low and high inocula of Trypanosoma cruzi in chagasic mice treated with nifurtimox and relation to immune response.

Reinfection of chronic chagasic mice after treatment with nifurtimox resulted in different outcomes according to the number of parasites used for inoculation. Nifurtimox-treated chagasic animals injected with 2,500 trypomastigotes developed higher parasitemia and increased mortality compared with nontreated chagasic mice. When reinfection was done with 25 trypomastigotes, treated and nontreated animals showed similar parasitemias and mortalities, which were significantly higher in nonchagasic controls infected for the first time. Immunological studies showed that treatment with nifurtimox led to a decrease in anti-Trypanosoma cruzi antibodies engaged in parasite destruction, inducing either complement-dependent lysis or antibody-dependent cytotoxicity, but no difference in anti-T. cruzi cell-mediated immunity was found between treated and nontreated chagasic animals. It is concluded that treatment with nifurtimox leads to a loss of resistance to reinfection with a large number of trypanosomes, which is maintained with challenge with a few parasites, and that these two thresholds of premunition are probably associated with humoral and cell-mediated anti-T. cruzi immune responses, respectively.

Induction of heart alterations by immunization with subcellular fractions from Crithidia fasciculata.

Immunization of mice with subcellular fractions ofC. fasciculata led to myocarditis and electrocardiographic alterations similar to those induced by immunization withT. cruzi, the etiological agent of Chagas disease, suggesting the presence of similar cardiotoxic antigens in both trypanosomatid flagellates.