Call for Consensus in the Evaluation of Circulating Matrix Metalloproteinases in Chagas Disease.

Infection with the Trypanosoma cruzi parasite is endemic in parts of America. Approximately 30% of people infected develop Chagas cardiomyopathy, the most common cause of heart failure in these regions. No suitable biomarker that reflects the evolution of the disease has been widely accepted as of yet. There is substantial evidence, however, of a strong inflammatory reaction following infection with T. cruzi that could activate matrix metalloproteinases (MMPs). Emerging research suggests the involvement of MMPs in Chagas cardiomyopathy and there is a growing interest in measuring the blood levels of MMPs as diagnostic and/or prognostic indicators of heart damage in Chagas patients. This perspective discusses the lack of consensus on the best method for MMP evaluation. Some studies are based on MMP concentrations and activities in serum whereas others use plasma. We believe that these different methods of evaluation have led to incongruent and poorly comparable data on the blood levels of MMPs in Chagas patients. A standard for the preparation of blood samples needs to be adopted for the study of MMPs as markers of Chagas cardiomyopathy to ensure better comparability of research results.

Characterization and Diagnostic Application of Trypanosoma cruzi Trypomastigote Excreted-Secreted Antigens Shed in Extracellular Vesicles Released from Infected Mammalian Cells.

Chagas disease, caused by Trypanosoma cruzi, although endemic in many parts of Central and South America, is emerging as a global health threat through the potential contamination of blood supplies. Consequently, in the absence of a gold standard assay for the diagnosis of Chagas disease, additional antigens or strategies are needed. A proteomic analysis of the trypomastigote excreted-secreted antigens (TESA) associated with exosomal vesicles shed by T. cruzi identified ∼80 parasite proteins, with the majority being trans-sialidases. Mass spectrometry analysis of immunoprecipitation products performed using Chagas immune sera showed a marked enrichment in a subset of TESA proteins. Of particular relevance for diagnostic applications were the retrotransposon hot spot (RHS) proteins, which are absent in Leishmania spp., parasites that often confound diagnosis of Chagas disease. Interestingly, serological screens using recombinant RHS showed a robust immunoreactivity with sera from patients with clinical stages of Chagas ranging from asymptomatic to advance cardiomyopathy and this immunoreactivity was comparable to that of crude TESA. More importantly, no cross-reactivity with RHS was detected with sera from patients with malaria, leishmaniasis, toxoplasmosis, or African sleeping sickness, making this protein an attractive reagent for diagnosis of Chagas disease.

Matrix metalloproteinases 2 and 9 as diagnostic markers in the progression to Chagas cardiomyopathy.

BACKGROUND: Infection with the Trypanosoma cruzi parasite is endemic in parts of Central and South America. Approximately 30% of those infected develop Chagas cardiomyopathy, the most common cause of heart failure in this region. No suitable biomarker is available that reflects the evolution of the disease. Although there is substantial evidence of a strong inflammatory reaction following infection that could activate matrix metalloproteinases (MMPs), their role in the development of Chagas cardiomyopathy is unknown. METHODS: A cross-sectional study was conducted in Bucaramanga, Colombia, from 2002 to 2006, including 144 patients at different stages of Chagas disease and 44 control patients. The potential enzyme activities of MMP-2 and MMP-9 in plasma samples were determined by gelatin zymography. Clinical data including T cruzi serology, electrocardiograms, and echocardiograms were recorded for all patients. RESULTS: Densitometric analysis of potential enzyme activities in plasma samples showed a significant increase of 72-kd MMP-2 (P < .001) and 92-kd MMP-9 (P < .001) in T cruzi seropositive patients compared with control subjects. Matrix metalloproteinase 9 showed significantly increased activity in patients with abnormal electrocardiogram (P < .004) and with dilated cardiomyopathy compared (P < .001) with controls. Analysis of the MMP-2 and MMP-9 results in relation to clinical data revealed that abnormal heart relaxation correlated positively with high MMP-2 levels in patients with dilated cardiomyopathy (r = 0.75, P < .01). CONCLUSIONS: Plasma MMP-2 and MMP-9 both appear to be useful biomarkers for detecting the advent and progression of cardiomyopathy in T cruzi-infected individuals.

B effector cells activated by a chimeric protein consisting of IL-2 and the ectodomain of TGF-ß receptor II induce potent antitumor immunity.

We have previously shown that interleukin (IL)-2 receptor-expressing lymphoid cells stimulated with a chimeric protein linking IL-2 to the ectodomain of TGF-ß receptor II (also known as FIST) become resistant to TGF-ß-mediated suppression and produce significant amounts of proinflammatory cytokines. In this study, we have characterized the antigen presentation properties of FIST-stimulated B cells (hereafter inducible B effector cells, iBEC). FIST converts naïve splenic B cells to B effector cells characterized by potent antigen presentation properties and production of TNFα and IFNγ. iBECs display hyperphosphorylation of STAT3 and STAT5 downstream of the IL-2 receptor and upregulation of T-bet expression. iBECs maintain B-cell identity based on the expression of PAX5 and CD19 and overexpress Smad7, which confers resistance to TGF-ß-mediated suppression of B-cell activation. iBEC antitumor immunity was determined by a mouse model of lymphoma-expressing ovalbumin (E.G7-OVA) as a specific tumor antigen. OVA-pulsed iBECs function as antigen-presenting cells (APC) in vitro by inducing the activation of OVA-specific CD4(+) and CD8(+) T cells, respectively, and in vivo by conferring complete protective immunity against E.G7-OVA tumor challenge. In addition, OVA-pulsed iBECs promote tumor regression in immunocompetent C57Bl/6 mice bearing E.G7-OVA tumors. In conclusion, iBECs represent an entirely novel B cell-derived APC for immune therapy of cancer.

Novel TGF-beta antagonist inhibits tumor growth and angiogenesis by inducing IL-2 receptor-driven STAT1 activation.

Carcinoma derived TGF-ß acts as a potent pro-oncogenic factor and suppresses antitumor immunity. To antagonize TGF-ß-mediated effects in tandem with a proinflammatory immune stimulus, we generated a chimeric protein borne of the fusion of IL-2 and the soluble extracellular domain of TGF-ßR II (FIST). FIST acts as a decoy receptor trapping active TGF-ß in solution and interacts with IL-2-responsive lymphoid cells, inducing a distinctive hyperactivation of STAT1 downstream of IL-2R, which in turn promotes SMAD7 overexpression. Consequently, FIST-stimulated lymphoid cells are resistant to TGF-ß-mediated suppression and produce significant amounts of proinflammatory cytokines. STAT1 hyperactivation further induces significant secretion of angiostatic CXCL10. Moreover, FIST upregulates T-bet expression in NK cells promoting a potent Th1-mediated antitumor response. As a result, FIST stimulation completely inhibits pancreatic cancer (PANC02) and melanoma (B16) tumor growth in immunocompetent C57BL/6 mice. In addition, melanoma cells expressing FIST fail to form tumors in CD8(-/-), CD4(-/-), B cell-deficient (µMT), and beige mice, but not in NOD-SCID and Rag2/γc knockout mice, consistent with the pivotal role of FIST-responsive, cancer-killing NK cells in vivo. In summary, FIST constitutes a novel strategy of treating cancer that targets both the host's angiogenic and innate immune response to malignant cells.

The human ortholog of granulocyte macrophage colony-stimulating factor and interleukin-2 fusion protein induces potent ex vivo natural killer cell activation and maturation.

Natural killer (NK) cells are appealing cellular pharmaceuticals for cancer therapy because of their innate ability to recognize and kill tumor cells. Therefore, the development of methods that can enhance the potency in their anticancer effect would be desirable. We have previously shown that a murine granulocyte macrophage colony-stimulating factor (GM-CSF)/interleukin 2 (IL-2) fusion protein displays novel antitumor properties in vivo compared with both cytokines in combination due to recruitment of NK cells. In the present work, we have found that human ortholog of the GM-CSF/IL-2 fusion protein (a.k.a. hGIFT2) induces robust NK cell activation ex vivo with significant secretion of RANTES and a 37-fold increase in IFNgamma production when compared with either IL-2 or GM-CSF single cytokine treatment or their combination. Moreover, hGIFT2 upregulates the expression of NK cell activating receptors NKp44, NKp46, and DNAM-1 (CD226), as well as CD69, CD107a, and IL-2Rbeta expression. In addition, hGIFT2 promotes NK cell maturation, based on the downregulation of CD117 expression and upregulation of CD11b. This phenotype correlates with significantly greater cytotoxicity against tumor cells. At the molecular level, hGIFT2 leads to a potent activation of Janus-activated kinases (JAK) downstream of both IL-2 and GM-CSF receptors (JAK1 and JAK2, respectively) and consequently leads to a hyperphosphorylation of signal transducers and activators of transcription (STAT)1, STAT3, and STAT5. In conclusion, hGIFT2 fusokine possesses unique biochemical properties distinct from IL-2 and GM-CSF, constitutes a novel and potent tool for ex vivo NK cell activation and maturation, and may be of use for cancer cell immunotherapy.

Matrix metalloproteinase-2 degrades the cytoskeletal protein alpha-actinin in peroxynitrite mediated myocardial injury.

Matrix metalloproteinase (MMP)-2 mediates myocardial ischemia-reperfusion injury which is characterized by enhanced peroxynitrite biosynthesis during early reperfusion. Direct infusion of peroxynitrite into isolated hearts activates MMP-2 prior to the loss in mechanical function. The mechanical dysfunction is prevented by MMPs inhibitors. MMP-2 is also found in the sarcomere of cardiomyocytes where it cleaves troponin I and myosin light chain I. Cytoskeletal proteins such as alpha-actinin, desmin and spectrin are found in close association with the sarcomere and are known to be degraded in ischemia-reperfusion injury. It remains unknown whether these proteins are degraded in peroxynitrite-induced myocardial injury and if cytoskeletal proteins are also targets for MMP-2. Peroxynitrite (80 microM) was infused into isolated rat hearts which led to a delayed onset but rapidly developing decline in mechanical function. The MMPs inhibitor PD-166793 or the peroxynitrite scavenger glutathione prevented the decline in cardiac function. At the end of perfusion, alpha-actinin levels were decreased by 45+/-3% in peroxynitrite-infused hearts as compared to control hearts; however, this was normalized to that of control hearts with either PD-166793 or glutathione. Cardiac desmin and alphaII spectrin levels were unaltered following peroxynitrite infusion. alpha-Actinin and to a lesser extent desmin are susceptible to in vitro proteolysis by MMP-2 whereas spectrin is resistant. Cardiac dysfunction induced by peroxynitrite involves degradation of alpha-actinin that may be mediated by the proteolytic action of MMP-2.

Hydrogen peroxide causes cardiac dysfunction independent from its effects on matrix metalloproteinase-2 activation.

Hydrogen peroxide (H2O2) causes cardiac dysfunction through multiple mechanisms. As oxidative stress can activate matrix metalloproteinases (MMPs) and, in particular, MMP-2 activity is associated with oxidative stress injury in the heart, we hypothesized that MMP-2 activation by H2O2 in isolated rat hearts contributes to cardiac dysfunction in this model. Isolated working rat hearts were perfused at 37 degrees C with a recirculating Krebs-Henseleit buffer+/-5 mmol/L pyruvate, known to protect hearts from oxidative stress. H2O2 (300 micromol/L) was added as a single bolus after 20 min of equilibration, and cardiac function was monitored for 60 min. MMPs activities in both the heart and perfusate samples were assessed by gelatin zymography. Tissue high energy phosphates were analysed by HPLC. The actions of 2 MMP inhibitors, doxycycline (75 micromol/L) or Ro 31-9790 (3 micromol/L), were also assessed. H2O2 at 300 micromol/L produced a rapid decline in cardiac mechanical function, which was maximal at 5 min. A peak in perfusate MMP-2 activity was also observed at 5 min. The deleterious effect of H2O2 on cardiac function was abolished by pyruvate but not by the MMPs inhibitors. This study suggests that in intact hearts, H2O2 induces contractile dysfunction independent of MMPs activation.

CD4 T cells play major effector role and CD8 T cells initiating role in spontaneous autoimmune myocarditis of HLA-DQ8 transgenic IAb knockout nonobese diabetic mice.

In humans, spontaneous autoimmune attack against cardiomyocytes often leads to idiopathic dilated cardiomyopathy (IDCM) and life-threatening heart failure. HLA-DQ8 transgenic IAb knockout NOD mice (NOD.DQ8/Ab(0); DQA1*0301, DQB1*0302) develop spontaneous anticardiomyocyte autoimmunity with pathology very similar to human IDCM, but why the heart is targeted is unknown. In the present study, we first investigated whether NOD/Ab(0) mice transgenic for a different DQ allele, DQ6, (DQA1*0102, DQB1*0602) would also develop myocarditis. NOD.DQ6/Ab(0) animals showed no cardiac pathology, implying that DQ8 is specifically required for the myocarditis phenotype. To further characterize the cellular immune mechanisms, we established crosses of our NOD.DQ8/Ab(0) animals with Rag1 knockout (Rag1(0)), Ig H chain knockout (IgH(0)), and beta(2)-microglobulin knockout (beta(2)m(0)) lines. Adoptive transfer of purified CD4 T cells from NOD.DQ8/Ab(0) mice with complete heart block (an indication of advanced myocarditis) into younger NOD.DQ8/Ab(0) Rag1(0) animals induced cardiac pathology in all recipients, whereas adoptive transfer of purified CD8 T cells or B lymphocytes had no effect. Despite the absence of B lymphocytes, NOD.DQ8/Ab(0)IgH(0) animals still developed complete heart block, whereas NOD.DQ8/Ab(0)beta(2)m(0) mice (which lack CD8 T cells) failed to develop any cardiac pathology. CD8 T cells (and possibly NK cells) seem to be necessary to initiate disease, whereas once initiated, CD4 T cells alone can orchestrate the cardiac pathology, likely through their capacity to recruit and activate macrophages. Understanding the cellular immune mechanisms causing spontaneous myocarditis/IDCM in this relevant animal model will facilitate the development and testing of new therapies for this devastating disease.

Autoimmune cardiomyopathy and heart block develop spontaneously in HLA-DQ8 transgenic IAbeta knockout NOD mice.

A line of nonobese diabetic (NOD) mice expressing the human diabetes-associated HLA-DQ8 transgene in the absence of mouse IAbeta failed to show spontaneous insulitis or diabetes, but rather developed dilated cardiomyopathy, leading to early death from heart failure. Pathology in these animals results from an organ- and cell-specific autoimmune response against normal cardiomyoctes in the atrial and ventricular walls, as well as against very similar myocytes present in the outermost muscle layer surrounding the pulmonary veins. Progression of the autoimmune process could be followed by serial ECG measurements; irradiation of young animals significantly delayed disease progression, and this effect could be reversed by adoptive transfer of splenocytes taken from older animals with complete heart block. Disease progression could also be blocked by cyclosporin A treatment, but was accelerated by injection of complete Fruend's adjuvant. The constellation of findings of spontaneously arising destructive focal lymphocytic infiltrates within the myocardium, rising titers of circulating anticardiac autoantibodies, dilation of the cardiac chambers, and gradual progression to end-stage heart failure bears a striking resemblance to what is seen in humans with idiopathic dilated cardiomyopathy, a serious and often life-threatening medical condition. This transgenic strain provides a highly relevant animal model for human autoimmune myocarditis and postinflammatory dilated cardiomyopathy.