Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates.

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in hospitals and research laboratories. In addition, the Trypanosoma cruzi infection is transmitted congenitally from a chagasic mother to her offspring, but the male partner's contribution to in utero contamination is unknown. The findings of nests and clumps of amastigotes and of trypomastigotes in the theca cells of the ovary, in the goniablasts and in the lumen of seminiferous tubules suggest that T. cruzi infections are sexually transmitted. The research protocol herein presents the results of a family study population showing parasite nuclear DNA in the diploid blood mononuclear cells and in the haploid gametes of human subjects. Thus, three independent biological samples collected one year apart confirmed that T. cruzi infections were sexually transmitted to progeny. Interestingly, the specific T. cruzi antibody was absent in the majority of family progeny that bore immune tolerance to the parasite antigen. Immune tolerance was demonstrated in chicken refractory to T. cruzi after the first week of embryonic growth, and chicks hatched from the flagellate-inoculated eggs were unable to produce the specific antibody. Moreover, the instillation of the human semen ejaculates intraperitoneally or into the vagina of naive mice yielded T. cruzi amastigotes in the epididymis, seminiferous tubule, vas deferens and uterine tube with an absence of inflammatory reactions in the immune privileged organs of reproduction. The breeding of T. cruzi-infected male and female mice with naive mates resulted in acquisition of the infections, which were later transmitted to the progeny. Therefore, a robust education, information and communication program that involves the population and social organizations is deemed necessary to prevent Chagas disease.

American trypanosomiasis and Chagas disease: Sexual transmission.

OBJECTIVE: To contribute to the discussion on the research findings indicating the sexual transmission of American trypanosomiasis and Chagas disease in humans. METHODS: A review of the literature was performed to investigate the routes of transmission of Trypanosoma cruzi parasites and to evaluate the distribution of Chagas disease, which is now found across five continents. RESULTS: The epidemiological profile of American trypanosomiasis, which is still considered a neglected disease of the poor people of Latin America, has changed over time. A family-based study demonstrated that the blood protozoan T. cruzi can be transmitted sexually from infected males and females to naïve mates. CONCLUSIONS: Evidence that Chagas disease can be transmitted sexually, coupled with the migration of individuals with Chagas disease to previously non-endemic countries and increased travel to endemic countries, has implications for public health. Improved screening of blood supplies and prenatal care are required to prevent congenital spread.

Low-Power Laser Irradiation (LPLI): A Clinical Point of View on a Promising Strategy to Improve Liver Regeneration.

The capacity of the liver to regenerate is an important clinical issue after major hepatectomies and makes the difference between life and death in some cases of post-operative malfunction when the liver remnant is too small or has an impaired regenerative capacity. Several approaches have been tested to stimulate hepatic regeneration after post-operative hepatic failure syndrome; however, they have produced controversial results. A quick, simple, and harmless method that can be used intraoperatively and capable of promoting an increased regenerative capacity of the remaining liver would be very welcome. Thus, based on the data in the literature, we presented low-power laser irradiation (LPLI) as a quick, simple, and harmless method to improve liver regeneration after major hepatectomies. This article highlights the current evidence about the effects of LPLI on liver regeneration, and also suggests laser therapy as an important tool for regenerative stimulation in clinical practice.

Low-power laser irradiation fails to improve liver regeneration in elderly rats at 48 h after 70 % resection.

The liver regeneration is an important clinical issue after major hepatectomies. Unfortunately, many organs (including the liver) exhibit age-related impairments regarding their regenerative capacity. Recent studies found that low-power laser irradiation (LPLI) has a stimulatory effect on the liver regeneration process. However, its effects in elderly remain unknown. Thus, this study aimed to investigate the main molecular mechanisms involved in liver regeneration of partially hepatectomized elderly rats exposed to LPLI. The effects of 15 min of LPLI (wavelength of 632.8 nm; fluence of 0.97 J/cm(2); total energy delivered of 3.6 J) were evaluated in hepatectomized elderly Wistar male rats. Afterwards, through immunoblotting approaches, the protein expression and phosphorylation levels of hepatocyte growth factor (HGF), Met, Akt and Erk 1/2 signaling pathways as well as the proliferating cell nuclear antigen (PCNA) were investigated. It was observed that LPLI was not able to improve liver regeneration in elderly rats as evidenced by the lack of improvement of HGF and PCNA protein expression or phosphorylation levels of Met, Akt and Erk 1/2 in the remnant livers. In sum, this study demonstrated that the main molecular pathway, i.e. HGF/Met → Akt and Erk 1/2 → PCNA, involved in the hepatic regeneration process was not improved by LPLI in elderly hepatectomized rats, which in turn rules out LPLI as an adjuvant therapy, as observed in this protocol of liver regeneration evaluation (i.e. at 48 h after 70 % resection), in elderly.

Inhibition of autoimmune Chagas-like heart disease by bone marrow transplantation.

BACKGROUND: Infection with the protozoan Trypanosoma cruzi manifests in mammals as Chagas heart disease. The treatment available for chagasic cardiomyopathy is unsatisfactory. METHODS/PRINCIPAL FINDINGS: To study the disease pathology and its inhibition, we employed a syngeneic chicken model refractory to T. cruzi in which chickens hatched from T. cruzi inoculated eggs retained parasite kDNA (1.4 kb) minicircles. Southern blotting with EcoRI genomic DNA digests revealed main 18 and 20 kb bands by hybridization with a radiolabeled minicircle sequence. Breeding these chickens generated kDNA-mutated F1, F2, and F3 progeny. A targeted-primer TAIL-PCR (tpTAIL-PCR) technique was employed to detect the kDNA integrations. Histocompatible reporter heart grafts were used to detect ongoing inflammatory cardiomyopathy in kDNA-mutated chickens. Fluorochromes were used to label bone marrow CD3+, CD28+, and CD45+ precursors of the thymus-dependent CD8α+ and CD8ß+ effector cells that expressed TCRγδ, vß1 and vß2 receptors, which infiltrated the adult hearts and the reporter heart grafts. CONCLUSIONS/SIGNIFICANCE: Genome modifications in kDNA-mutated chickens can be associated with disruption of immune tolerance to compatible heart grafts and with rejection of the adult host's heart and reporter graft, as well as tissue destruction by effector lymphocytes. Autoimmune heart rejection was largely observed in chickens with kDNA mutations in retrotransposons and in coding genes with roles in cell structure, metabolism, growth, and differentiation. Moreover, killing the sick kDNA-mutated bone marrow cells with cytostatic and anti-folate drugs and transplanting healthy marrow cells inhibited heart rejection. We report here for the first time that healthy bone marrow cells inhibited heart pathology in kDNA+ chickens and thus prevented the genetically driven clinical manifestations of the disease.

Liver regeneration following partial hepatectomy is improved by enhancing the HGF/Met axis and Akt and Erk pathways after low-power laser irradiation in rats.

A simple, easy, and safe procedure aiming to improve liver regeneration could be of great clinical benefit in critical situations such as major hepatectomy, trauma, or hemorrhage. Low-power laser irradiation (LPLI) has come into a wide range of use in clinical practice by inducing regeneration in healthy and injured tissues. However, the effect of LPLI on the process of liver regeneration, especially those related to the molecular mechanisms, is not fully understood. Thus, the aim of the present study was to investigate the main molecular mechanisms involved in liver regeneration of partially hepatectomized rats exposed to LPLI. We used Wistar male rats, which had their remaining liver irradiated or not with LPLI (wavelength of 632.8 nm and fluence of 65 mW/cm(2)) for 15 min after a 70% hepatectomy. We subsequently investigated hepatocyte growth factor (HGF), Met, Akt, and Erk 1/2 signaling pathways through protein expression and phosphorylation analyses along with cell proliferation (proliferating cell nuclear antigen (PCNA) and Ki-67) using immunoblotting and histological studies. Our results show that LPLI can improve liver regeneration as shown by increased HGF protein expression and the phosphorylation levels of Met, Akt, and Erk 1/2 accompanied by higher levels of the PCNA and Ki-67 protein in the remnant livers. In summary, our results suggest that LPLI may play a clinical role as a simple, fast, and easy-to-perform strategy in order to enhance the liver regenerative capacity of a small liver remnant after hepatectomy.

Parasite induced genetically driven autoimmune Chagas heart disease in the chicken model.

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases show Chagas disease up to three decades or later. Autoimmunity and parasite persistence are competing theories to explain the pathogenesis of Chagas disease. To separate roles played by parasite persistence and autoimmunity in Chagas disease we inoculate the T. cruzi in the air chamber of fertilized eggs. The mature chicken immune system is a tight biological barrier against T. cruzi and the infection is eradicated upon development of its immune system by the end of the first week of growth. The chicks are parasite-free at hatching, but they retain integrated parasite mitochondrial kinetoplast DNA (kDNA) minicircle within their genome that are transferred to their progeny. Documentation of the kDNA minicircle integration in the chicken genome was obtained by a targeted prime TAIL-PCR, Southern hybridizations, cloning, and sequencing. The kDNA minicircle integrations rupture open reading frames for transcription and immune system factors, phosphatase (GTPase), adenylate cyclase and phosphorylases (PKC, NF-Kappa B activator, PI-3K) associated with cell physiology, growth, and differentiation, and other gene functions. Severe myocarditis due to rejection of target heart fibers by effectors cytotoxic lymphocytes is seen in the kDNA mutated chickens, showing an inflammatory cardiomyopathy similar to that seen in human Chagas disease. Notably, heart failure and skeletal muscle weakness are present in adult chickens with kDNA rupture of the dystrophin gene in chromosome 1. Similar genotipic alterations are associated with tissue destruction carried out by effectors CD45+, CD8γδ+, CD8α lymphocytes. Thus this protozoan infection can induce genetically driven autoimmune disease.

Interactome: Smart hematophagous triatomine salivary gland molecules counteract human hemostasis during meal acquisition.

Human populations are constantly plagued by hematophagous insects' bites, in particular the triatomine insects that are vectors of the Trypanosoma cruzi agent in Chagas disease. The pharmacologically-active molecules present in the salivary glands of hematophagous insects are injected into the human skin to initiate acquisition of blood meals. Sets of vasodilators, anti-platelet aggregators, anti-coagulants, immunogenic polypeptides, anesthetics, odorants, antibiotics, and detoxifying molecules have been disclosed with the aid of proteomics and recombinant cDNA techniques. These molecules can provide insights about the insect-pathogen-host interactions essential for understanding the physiopathology of the insect bite. The data and information presented in this review aim for the development of new drugs to prevent insect bites and the insect-transmitted endemic of Chagas disease.

Real-time PCR in HIV/Trypanosoma cruzi coinfection with and without Chagas disease reactivation: association with HIV viral load and CD4 level.

BACKGROUND: Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. METHODOLOGY: Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. PRINCIPAL FINDINGS: qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4(+) cells or the CD4(+)/CD8(+) ratio. CONCLUSIONS: qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4(+) cells/mm(3) and the CD4(+)/CD8(+) ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.

Pathogenesis of chagas' disease: parasite persistence and autoimmunity.

Acute Trypanosoma cruzi infections can be asymptomatic, but chronically infected individuals can die of Chagas' disease. The transfer of the parasite mitochondrial kinetoplast DNA (kDNA) minicircle to the genome of chagasic patients can explain the pathogenesis of the disease; in cases of Chagas' disease with evident cardiomyopathy, the kDNA minicircles integrate mainly into retrotransposons at several chromosomes, but the minicircles are also detected in coding regions of genes that regulate cell growth, differentiation, and immune responses. An accurate evaluation of the role played by the genotype alterations in the autoimmune rejection of self-tissues in Chagas' disease is achieved with the cross-kingdom chicken model system, which is refractory to T. cruzi infections. The inoculation of T. cruzi into embryonated eggs prior to incubation generates parasite-free chicks, which retain the kDNA minicircle sequence mainly in the macrochromosome coding genes. Crossbreeding transfers the kDNA mutations to the chicken progeny. The kDNA-mutated chickens develop severe cardiomyopathy in adult life and die of heart failure. The phenotyping of the lesions revealed that cytotoxic CD45, CD8(+) γδ, and CD8α(+) T lymphocytes carry out the rejection of the chicken heart. These results suggest that the inflammatory cardiomyopathy of Chagas' disease is a genetically driven autoimmune disease.

Diversity of anti-haemostatic proteins in the salivary glands of Rhodnius species transmitters of Chagas disease in the greater Amazon.

The triatomines in the tribe Rhodniini are the main vectors of the Trypanosoma cruzi to humans in recent outbreaks of acute Chagas disease in the Amazon. These insects dwelling in palm trees do not colonize the human domicile. Their success to transmit the infection relies partially on the efficacy of their salivary gland apparatuses. Here we show the transcriptome of the Rhodnius brethesi and Rhodnius robustus salivary glands, comprising 56 and 122 clusters, respectively. Approximately one third of these clusters are described for the first time. The LC-MS/MS analysis identified 123 and 111 proteins in R. brethesi and R. robustus sialome, respectively. Noteworthy, lipocalin platelet aggregation inhibitors, inositol polyphosphate 5-phosphatases, and Kazal domain proteins, which are essential for the insect's successful acquisition of blood meals, were found in our analysis. Moreover, glutathione S transferase and antigen-5, which play roles in the insect's defense and resistance against insecticide, were also observed.

Redundancy of proteins in the salivary glands of Panstrongylus megistus secures prolonged procurement for blood meals.

Panstrongylus megistus, a vector for the Chagas disease parasite Trypanosoma cruzi, is a hematophagous bug widely distributed in South America. This ubiquitous triatomine is known to colonize different wild life habitats. Additionally, P. megistus synanthropy, preying upon mammals, birds, reptiles, and eventually being predators upon insect's hemolymph probably increases its ability to survive after prolonged fasting. It was suspected that the P. megistus mechanisms of adaptation to survival might include a salivary gland complex tool-box with a diversity of pharmacologically active proteins for obtaining blood meals. Herein we describe comprehensive proteome and transcriptome of the P. megistus salivary gland. The proteomic analysis led to the identification of 159 proteins, and the transcriptome revealed 47 complete cDNAs. A diversity of protein functions associated to blood feeding was identified. The most prevalent proteins were related to blood clotting, anti-platelet aggregation and anti-vasoconstriction activities, which correlate with the insect's ability to obtain meals from different sources. Moreover, a gene of resistance to insecticides was identified. These features augments the comprehension towards P. megistus enormous capacity to survive in adverse wild life-changing habitats.

Trypanosoma cruzi in the chicken model: Chagas-like heart disease in the absence of parasitism.

BACKGROUND: The administration of anti-trypanosome nitroderivatives curtails Trypanosoma cruzi infection in Chagas disease patients, but does not prevent destructive lesions in the heart. This observation suggests that an effective treatment for the disease requires understanding its pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: To understand the origin of clinical manifestations of the heart disease we used a chicken model system in which infection can be initiated in the egg, but parasite persistence is precluded. T. cruzi inoculation into the air chamber of embryonated chicken eggs generated chicks that retained only the parasite mitochondrial kinetoplast DNA minicircle in their genome after eight days of gestation. Crossbreeding showed that minicircles were transferred vertically via the germ line to chicken progeny. Minicircle integration in coding regions was shown by targeted-primer thermal asymmetric interlaced PCR, and detected by direct genomic analysis. The kDNA-mutated chickens died with arrhythmias, shortness of breath, cyanosis and heart failure. These chickens with cardiomyopathy had rupture of the dystrophin and other genes that regulate cell growth and differentiation. Tissue pathology revealed inflammatory dilated cardiomegaly whereby immune system mononuclear cells lyse parasite-free target heart fibers. The heart cell destruction implicated a thymus-dependent, autoimmune; self-tissue rejection carried out by CD45(+), CD8γδ(+), and CD8α lymphocytes. CONCLUSIONS/SIGNIFICANCE: These results suggest that genetic alterations resulting from kDNA integration in the host genome lead to autoimmune-mediated destruction of heart tissue in the absence of T. cruzi parasites.

2-DE-based proteomic investigation of the saliva of the Amazonian triatomine vectors of Chagas disease: Rhodnius brethesi and Rhodnius robustus.

The triatomine bugs are obligatory haematophagous organisms that act as vectors of Chagas disease by transmitting the protozoan Trypanosoma cruzi. Their feeding success is strongly related to salivary proteins that allow these insects to access blood by counteracting host haemostatic mechanisms. Proteomic studies were performed on saliva from the Amazonian triatomine bugs: Rhodnius brethesi and R. robustus, species epidemiologically relevant in the transmission of T. cruzi. Initially, salivary proteins were separated by two-dimensional gel electrophoresis (2-DE). The average number of spots of the R. brethesi and R. robustus saliva samples were 129 and 135, respectively. The 2-DE profiles were very similar between the two species. Identification of spots by peptide mass fingerprinting afforded limited efficiency, since very few species-specific salivary protein sequences are available in public sequence databases. Therefore, peptide fragmentation and de novo sequencing using a MALDI-TOF/TOF mass spectrometer were applied for similarity-driven identifications which generated very positive results. The data revealed mainly lipocalin-like proteins which promote blood feeding of these insects. The redundancy of saliva sequence identification suggested multiple isoforms caused by gene duplication followed by gene modification and/or post-translational modifications. In the first experimental assay, these proteins were predominantly phosphorylated, suggesting functional phosphoregulation of the lipocalins.

Inheritance of DNA transferred from American trypanosomes to human hosts.

Interspecies DNA transfer is a major biological process leading to the accumulation of mutations inherited by sexual reproduction among eukaryotes. Lateral DNA transfer events and their inheritance has been challenging to document. In this study we modified a thermal asymmetric interlaced PCR by using additional targeted primers, along with Southern blots, fluorescence techniques, and bioinformatics, to identify lateral DNA transfer events from parasite to host. Instances of naturally occurring human infections by Trypanosoma cruzi are documented, where mitochondrial minicircles integrated mainly into retrotransposable LINE-1 of various chromosomes. The founders of five families show minicircle integrations that were transferred vertically to their progeny. Microhomology end-joining of 6 to 22 AC-rich nucleotide repeats in the minicircles and host DNA mediates foreign DNA integration. Heterogeneous minicircle sequences were distributed randomly among families, with diversity increasing due to subsequent rearrangement of inserted fragments. Mosaic recombination and hitchhiking on retrotransposition events to different loci were more prevalent in germ line as compared to somatic cells. Potential new genes, pseudogenes, and knockouts were identified. A pathway of minicircle integration and maintenance in the host genome is suggested. Thus, infection by T. cruzi has the unexpected consequence of increasing human genetic diversity, and Chagas disease may be a fortuitous share of negative selection. This demonstration of contemporary transfer of eukaryotic DNA to the human genome and its subsequent inheritance by descendants introduces a significant change in the scientific concept of evolutionary biology and medicine.

Environment, interactions between Trypanosoma cruzi and its host, and health.

An epidemiological chain involving Trypanosoma cruzi is discussed at the environmental level, and in terms of fine molecular interactions in invertebrate and vertebrate hosts dwelling in different ecosystems. This protozoan has a complex, genetically controlled plasticity, which confers adaptation to approximately 40 blood-sucking triatomine species and to over 1,000 mammalian species, fulfilling diverse metabolic requirements in its complex life-cycle. The Tr. cruzi infections are deeply embedded in countless ecotypes, where they are difficult to defeat using the control methods that are currently available. Many more field and laboratory studies are required to obtain data and information that may be used for the control and prevention of Tr. cruzi infections and their various disease manifestations. Emphasis should be placed on those sensitive interactions at cellular and environmental levels that could become selected targets for disease prevention. In the short term, new technologies for social mobilization should be used by people and organizations working for justice and equality through health information and promotion. A mass media directed program could deliver education, information and communication to protect the inhabitants at risk of contracting Tr. cruzi infections.

Environment, interactions between Trypanosoma cruzi and its host, and health: [review] / Meio-ambiente, interações entre Trypanosoma cruzi e seu hospedeiro e saúde humana: [revisão]

An epidemiological chain involving Trypanosoma cruzi is discussed at the environmental level, and in terms of fine molecular interactions in invertebrate and vertebrate hosts dwelling in different ecosystems. This protozoan has a complex, genetically controlled plasticity, which confers adaptation to approximately 40 blood-sucking triatomine species and to over 1,000 mammalian species, fulfilling diverse metabolic requirements in its complex life-cycle. The Tr. cruzi infections are deeply embedded in countless ecotypes, where they are difficult to defeat using the control methods that are currently available. Many more field and laboratory studies are required to obtain data and information that may be used for the control and prevention of Tr. cruzi infections and their various disease manifestations. Emphasis should be placed on those sensitive interactions at cellular and environmental levels that could become selected targets for disease prevention. In the short term, new technologies for social mobilization should be used by people and organizations working for justice and equality through health information and promotion. A mass media directed program could deliver education, information and communication to protect the inhabitants at risk of contracting Tr. cruzi infections.

Uma rede epidemiológica envolvendo o Trypanosoma cruzi foi discutida nos níveis ambientais e de interações moleculares nos hospedeiros que habitam em 19 diferentes ecossistemas. O protozoário tem uma enorme plasticidade controlada geneticamente que confere sua adaptação a cerca de quarenta espécies de triatomíneos e mais de mil espécies de mamíferos. Essas infecções estão profundamente embutidas em inúmeros ecótopos, onde elas estão inacessíveis aos métodos de controle utilizados. Muito mais estudos de campo e de laboratório são necessários à obtenção de dados e informação pertinentes ao controle e prevenção das infecções pelo Tr. cruzi e as várias manifestações da doença. Ênfase deve ser dada àquelas interações que ocorrem nos níveis celulares e ambientais que se poderiam tomar como alvos seletivos para prevenção da doença. Novas tecnologias para mobilização social devem ser disponibilizadas para os que trabalham pela justiça e pela igualdade, mediante informação para a promoção da saúde. Um programa direcionado de educação de massa pode prover informação e comunicação necessárias para proteger os habitantes atualmente expostos ao risco de contrair as infecções pelo Tr. cruzi.