TcI isolates of Trypanosoma cruzi exploit the antioxidant network for enhanced intracellular survival in macrophages and virulence in mice

Trypanosoma cruzi species is categorized into six discrete typing units (TcI to TcVI) of which TcI is most abundantly noted in the sylvatic transmission cycle and considered the major cause of human disease. In our study, the TcI strains Colombiana (COL), SylvioX10/4 (SYL), and a cultured clone (TCC) exhibited different biological behavior in a murine model, ranging from high parasitemia and symptomatic cardiomyopathy (SYL), mild parasitemia and high tissue tropism (COL), to no pathogenicity (TCC). Proteomic profiling of the insect (epimastigote) and infective (trypomastigote) forms by two-dimensional gel electrophoresis/ matrix-assisted laser desorption ionization-time of flight mass spectrometry, followed by functional annotation of the differential proteome data sets (≥2-fold change, P<0.05), showed that several proteins involved in (i) cytoskeletal assembly and remodeling, essential for flagellar wave frequency and amplitude and forward motility of the parasite, and (ii) the parasite-specific antioxidant network were enhanced in COL and SYL (versus TCC) trypomastigotes. Western blotting confirmed the enhanced protein levels of cytosolic and mitochondrial tryparedoxin peroxidases and their substrate (tryparedoxin) and iron superoxide dismutase in COL and SYL (versus TCC) trypomastigotes. Further, COL and SYL (but not TCC) were resistant to exogenous treatment with stable oxidants (H2O2 and peroxynitrite [ONOO-]) and dampened the intracellular superoxide and nitric oxide response in macrophages, and thus these isolates escaped from macrophages. Our findings suggest that protein expression conducive to increase in motility and control of macrophage-derived free radicals provides survival and persistence benefits to TcI isolates of T. cruzi.Fil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Hosakote, Yashoda M.. University of Texas Medical Branch; Estados UnidosFil: Koo, Sue Jie. University of Texas Medical Branch; Estados UnidosFil: Dhiman, Monisha. University of Texas Medical Branch; Estados UnidosFil: Piñeyro, María Dolores. Instituto Pasteur de Montevideo. Unidad de Biología Molecular; Uruguay. Universidad de la Republica; UruguayFil: Parodi­ Talice, Adriana. Instituto Pasteur de Montevideo. Unidad de Biología Molecular; Uruguay. Universidad de la Republica; UruguayFil: Basombrío, Miguel Ángel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Robello, Carlos. Instituto Pasteur de Montevideo. Unidad de Biología Molecular; Uruguay. Universidad de la Republica; UruguayFil: Garg, Nisha J.. University of Texas Medical Branch; Estados Unido

Defects of mtDNA replication impaired mitochondrial biogenesis during Trypanosoma cruzi infection in human cardiomyocytes and Chagasic patients: The role of Nrf1/2 and antioxidant response

Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)-regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi-infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)-regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi-infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for replication and gene expression in Chagas disease.Fil: Wan, Xianxiu. University of Texas Medical Branch; Estados UnidosFil: Gupta, Shivali. University of Texas Medical Branch; Estados UnidosFil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Davidson, Mercy M.. Columbia University; Estados UnidosFil: Dousset, Pierre. Hospital San Bernardo; ArgentinaFil: Amoroso, Alejandro. Hospital San Bernardo; ArgentinaFil: Garg, Nisha Jain. University of Texas Medical Branch; Estados Unido

A Monoallelic Deletion of the TcCRT Gene Increases the Attenuation of a Cultured Trypanosoma cruzi Strain, Protecting Against an in Vivo Virulent Challenge

Trypanosoma cruzi calreticulin (TcCRT) is a virulence factor that binds complement C1, thus inhibiting the activation of the classical complement pathway and generating pro-phagocytic signals that increase parasite infectivity. In a previous work, we characterized a clonal cell line lacking one TcCRT allele (TcCRT+/-) and another overexpressing it (TcCRT+), both derived from the attenuated TCC T. cruzi strain. The TcCRT+/- mutant was highly susceptible to killing by the complement machinery and presented a remarkable reduced propagation and differentiation rate both in vitro and in vivo. In this report, we have extended these studies to assess, in a mouse model of disease, the virulence, immunogenicity and safety of the mutant as an experimental vaccine. Balb/c mice were inoculated with TcCRT+/- parasites and followed-up during a 6-month period. Mutant parasites were not detected by sensitive techniques, even after mice immune suppression. Total anti-T. cruzi IgG levels were undetectable in TcCRT+/- inoculated mice and the genetic alteration was stable after long-term infection and it did not revert back to wild type form. Most importantly, immunization with TcCRT+/- parasites induces a highly protective response after challenge with a virulent T. cruzi strain, as evidenced by lower parasite density, mortality, spleen index and tissue inflammatory response. TcCRT+/- clones are restricted in two important properties conferred by TcCRT and indirectly by C1q: their ability to evade the host immune response and their virulence. Therefore, deletion of one copy of the TcCRT gene in the attenuated TCC strain generated a safe and irreversibly gene-deleted live attenuated parasite with high immunoprotective properties. Our results also contribute to endorse the important role of TcCRT as a T. cruzi virulence factor.Fil: Sánchez Valdéz, Fernando Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Perez Brandan, Cecilia Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Ramirez, Galia. Universidad de Santiago de Chile; ChileFil: Uncos, Delfor Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Cimino, Ruben O.. Universidad Nacional de Salta. Facultad de Cs.naturales. Escuela de Biologia; ArgentinaFil: Cardozo, Ruben M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Marco, Jorge Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Ferreira, Arturo. Universidad de Santiago de Chile; ChileFil: Basombrio, Miguel Angel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentin

A Monoallelic Deletion of the TcCRT Gene Increases the Attenuation of a Cultured Trypanosoma cruzi Strain, Protecting Against an in Vivo Virulent Challenge

Trypanosoma cruzi calreticulin (TcCRT) is a virulence factor that binds complement C1, thus inhibiting the activation of the classical complement pathway and generating pro-phagocytic signals that increase parasite infectivity. In a previous work, we characterized a clonal cell line lacking one TcCRT allele (TcCRT+/-) and another overexpressing it (TcCRT+), both derived from the attenuated TCC T. cruzi strain. The TcCRT+/- mutant was highly susceptible to killing by the complement machinery and presented a remarkable reduced propagation and differentiation rate both in vitro and in vivo. In this report, we have extended these studies to assess, in a mouse model of disease, the virulence, immunogenicity and safety of the mutant as an experimental vaccine. Balb/c mice were inoculated with TcCRT+/- parasites and followed-up during a 6-month period. Mutant parasites were not detected by sensitive techniques, even after mice immune suppression. Total anti-T. cruzi IgG levels were undetectable in TcCRT+/- inoculated mice and the genetic alteration was stable after long-term infection and it did not revert back to wild type form. Most importantly, immunization with TcCRT+/- parasites induces a highly protective response after challenge with a virulent T. cruzi strain, as evidenced by lower parasite density, mortality, spleen index and tissue inflammatory response. TcCRT+/- clones are restricted in two important properties conferred by TcCRT and indirectly by C1q: their ability to evade the host immune response and their virulence. Therefore, deletion of one copy of the TcCRT gene in the attenuated TCC strain generated a safe and irreversibly gene-deleted live attenuated parasite with high immunoprotective properties. Our results also contribute to endorse the important role of TcCRT as a T. cruzi virulence factor.Fil: Sánchez Valdéz, Fernando Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Perez Brandan, Cecilia Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Ramirez, Galia. Universidad de Santiago de Chile; ChileFil: Uncos, Delfor Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Cimino, Ruben O.. Universidad Nacional de Salta. Facultad de Cs.naturales. Escuela de Biologia; ArgentinaFil: Cardozo, Ruben M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Marco, Jorge Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Ferreira, Arturo. Universidad de Santiago de Chile; ChileFil: Basombrio, Miguel Angel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentin

Innate immune responses and antioxidant/oxidant imbalance are major determinants of human Chagas disease.

We investigated the pathological and diagnostic role of selected markers of inflammation, oxidant/antioxidant status, and cellular injury in human Chagas disease. METHODS: Seropositive/chagasic subjects characterized as clinically-symptomatic or clinically-asymptomatic (n = 116), seronegative/cardiac subjects (n = 102), and seronegative/healthy subjects (n = 45) were analyzed for peripheral blood biomarkers. RESULTS: Seropositive/chagasic subjects exhibited an increase in sera or plasma levels of myeloperoxidase (MPO, 2.8-fold), advanced oxidation protein products (AOPP, 56%), nitrite (5.7-fold), lipid peroxides (LPO, 12-17-fold) and malondialdehyde (MDA, 4-6-fold); and a decline in superoxide dismutase (SOD, 52%) and glutathione (GSH, 75%) contents. Correlation analysis identified a significant (p0.95). The MPO (r = 0.664) and LPO (r = 0.841) levels were also correlated with clinical disease state in chagasic subjects (p<0.001). Seronegative/cardiac subjects exhibited up to 77% decline in SOD, 3-5-fold increase in LPO and glutamate pyruvate transaminase (GPT) levels, and statistically insignificant change in MPO, AOPP, MDA, GPX, GSH, and creatine kinase (CK) levels. CONCLUSIONS: The interlinked effects of innate immune responses and antioxidant/oxidant imbalance are major determinants of human Chagas disease. The MPO, LPO and nitrite are excellent biomarkers for diagnosing seropositive/chagasic subjects, and MPO and LPO levels have potential utility in identifying clinical severity of Chagas diseaseFil: Dhiman, Monisha. University Of Texas Medical Branch. Department Of Microbiology & Immunology And Pathology; United State of America;Fil: Coronado, Yun A.. University Of Texas Medical Branch. Department Of Microbiology & Immunology And Pathology; United State of America;Fil: Vallejo, Cecilia K.. University Of Texas Medical Branch. Department Of Microbiology & Immunology And Pathology; United State of America;Fil: Petersen, John R.. University of Texas Medical Branch. Department of Pathology; United States of America;Fil: Ejilemele, Adetoum. University of Texas Medical Branch. Department of Pathology; United States of America;Fil: Nuñez, Sonia. Hospital Público de Gestión Descentralizada San Bernardo (HPGDSA); Argentina;Fil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Salta. Instituto de Patologia Experimental; Argentina;Fil: Spratt, Heidi. Departments of Biochemistry and Molecular Biology and Preventive Medicine and Community Health. University of Texas Medical Branch; United States of America;Fil: Garg, Nisha Jain. University of Texas Medical Branch. Department of Pathology; United States of America

Cardiac-Oxidized Antigens Are Targets of Immune Recognition by Antibodies and Potential Molecular Determinants in Chagas Disease Pathogenesis

Trypanosoma cruzi elicits reactive oxygen species (ROS) of inflammatory and mitochondrial origin in infected hosts. In this study, we examined ROS-induced oxidative modifications in the heart and determined whether the resultant oxidized cardiac proteins are targets of immune response and of pathological significance in Chagas disease. Heart biopsies from chagasic mice, rats and human patients exhibited, when compared to those from normal controls, a substantial increase in protein 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), carbonyl, and 3-nitrotyrosine (3-NT) adducts. To evaluate whether oxidized proteins gain antigenic properties, heart homogenates or isolated cardiomyocytes were oxidized in vitro and one- or two-dimensional gel electrophoresis (2D-GE)/Western blotting (WB) was performed to investigate the proteomic oxidative changes and recognition of oxidized proteins by sera antibodies in chagasic rodents (mice, rats) and human patients. Human cardiomyocytes exhibited LD50 sensitivity to 30 µM 4-HNE and 100 µM H2O2 at 6 h and 12 h, respectively. In vitro oxidation with 4-HNE or H2O2 resulted in a substantial increase in 4-HNE- and carbonyl-modified proteins that correlated with increased recognition of cardiac (cardiomyocytes) proteins by sera antibodies of chagasic rodents and human patients. 2D-GE/Western blotting followed by MALDI-TOF-MS/MS analysis to identify cardiac proteins that were oxidized and recognized by human chagasic sera yielded 82 unique proteins. We validated the 2D-GE results by enzyme-linked immunosorbent assay (ELISA) and WB and demonstrated that oxidation of recombinant titin enhanced its immunogenicity and recognition by sera antibodies from chagasic hosts (rats and humans). Treatment of infected rats with phenyl-α-tert-butyl nitrone (PBN, antioxidant) resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic rats. We conclude that ROS-induced, cardiac-oxidized antigens are targets of immune recognition by antibodies and molecular determinants for pathogenesis during Chagas disease

Heterogeneity of prodromal Parkinson symptoms in siblings of Parkinson disease patients

A prodromal phase of Parkinson’s disease (PD) may precede motor manifestations by decades. PD patients’ siblings are at higher risk for PD, but the prevalence and distribution of prodromal symptoms are unknown. The study objectives were (1) to assess motor and non-motor features estimating prodromal PD probability in PD siblings recruited within the European PROPAG-AGEING project; (2) to compare motor and non-motor symptoms to the well-established DeNoPa cohort. 340 PD siblings from three sites (Bologna, Seville, Kassel/Goettingen) underwent clinical and neurological evaluations of PD markers. The German part of the cohort was compared with German de novo PD patients (dnPDs) and healthy controls (CTRs) from DeNoPa. Fifteen (4.4%) siblings presented with subtle signs of motor impairment, with MDS-UPDRS-III scores not clinically different from CTRs. Symptoms of orthostatic hypotension were present in 47 siblings (13.8%), no different to CTRs (p = 0.072). No differences were found for olfaction and overall cognition; German-siblings performed worse than CTRs in visuospatial-executive and language tasks. 3/147 siblings had video-polysomnography-confirmed REM sleep behavior disorder (RBD), none was positive on the RBD Screening Questionnaire. 173/300 siblings had &lt;1% probability of having prodromal PD; 100 between 1 and 10%, 26 siblings between 10 and 80%, one fulfilled the criteria for prodromal PD. According to the current analysis, we cannot confirm the increased risk of PD siblings for prodromal PD. Siblings showed a heterogeneous distribution of prodromal PD markers and probability. Additional parameters, including strong disease markers, should be investigated to verify if these results depend on validity and sensitivity of prodromal PD criteria, or if siblings’ risk is not elevated

The WSES/SICG/ACOI/SICUT/AcEMC/SIFIPAC guidelines for diagnosis and treatment of acute left colonic diverticulitis in the elderly

Acute left colonic diverticulitis (ALCD) in the elderly presents with unique epidemiological features when compared with younger patients. The clinical presentation is more nuanced in the elderly population, having higher in-hospital and postoperative mortality. Furthermore, geriatric comorbidities are a risk factor for complicated diverticulitis. Finally, elderly patients have a lower risk of recurrent episodes and, in case of recurrence, a lower probability of requiring urgent surgery than younger patients. The aim of the present work is to study age-related factors that may support a unique approach to the diagnosis and treatment of this problem in the elderly when compared with the WSES guidelines for the management of acute left-sided colonic diverticulitis. During the 1 degrees Pisa Workshop of Acute Care & Trauma Surgery held in Pisa (Italy) in September 2019, with the collaboration of the World Society of Emergency Surgery (WSES), the Italian Society of Geriatric Surgery (SICG), the Italian Hospital Surgeons Association (ACOI), the Italian Emergency Surgery and Trauma Association (SICUT), the Academy of Emergency Medicine and Care (AcEMC) and the Italian Society of Surgical Pathophysiology (SIFIPAC), three panel members presented a number of statements developed for each of the four themes regarding the diagnosis and management of ALCD in older patients, formulated according to the GRADE approach, at a Consensus Conference where a panel of experts participated. The statements were subsequently debated, revised, and finally approved by the Consensus Conference attendees. The current paper is a summary report of the definitive guidelines statements on each of the following topics: diagnosis, management, surgical technique and antibiotic therapy.Peer reviewe

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

Immunity and vaccine development efforts against Trypanosoma cruzi

Artículo de revisión especializadoTrypanosoma cruzi (T. cruzi) is the causative agent for Chagas disease (CD). There is a critical lack of methods for prevention of infection or treatment of acute infection and chronic disease. Studies in experimental models have suggested that the protective immunity against T. cruzi infection requires the elicitation of Th1 cytokines, lytic antibodies and the concerted activities of macrophages, T helper cells, and cytotoxic T lymphocytes (CTLs). In this review, we summarize the research efforts in vaccine development to date and the challenges faced in achieving an efficient prophylactic or therapeutic vaccine against human CD.UTM