Phosphorus protects cardiac tissue by modifying the immune response in rats infected by Trypanosoma cruzi.

AIM: This study evaluates and correlates the number of myocarditis focuses and production of cytokines in Rattus norvegicus (Wistar lineage), experimentally infected with T. Cruzi and treated with Phosphorus. METHODS: In two blind, controlled and randomized trials, 53 45-day-old, male animals were allocated into groups Control (n=24): Control group infected and treated with 7% hydroalcoholic solution, the preparation vehicle of the test medication; and Phosphorus (n=24 on days 0, 5, 10 and 24 after infection): group infected and treated with Phosphorus 13cH, diluted 10-26 and dynamized (test medication). The animals were inoculated intraperitoneally with 5×106 blood trypomastigotes of T. cruzi-Y strain. The medication was administered overnight (16 consecutive hours), diluted in water (1mL/100mL) in amber water bottles. The animals were treated 2days before and 2, 4, and 6days after infection. Enumeration of inflammatory foci in cardiac tissue (Hematoxylin-Eosin) and dosage of cytokines TNF-α and IFN-γ in the serum were performed on days 0, 5, 10 and 24 after infection, using three animals/group. Mann-Whitney, Friedman ANOVA, Spearman correlation (p<0.05), and Statistica Single User Software version 13.2 were used for data analysis. RESULTS: The animals treated with Phosphorus 13cH had high concentration of INF-É£ on the 5th day of infection with significant decrease on the 10th and 24th days (p<0.05), and high concentration of TNF-α on the 5th and 10th days of infection with decrease on the 24th day (p<0.05). The treatment with Phosphorus caused a significant increase of INF-É£ and TNF-α on the 5th day of infection compared with the Control (p<0.05), with reestablishment on the 24th day, as well as in the Control group. The group treated with Phosphorus had 52.5% less number of myocarditis focuses in heart than Control group (p<0.05) on the 10th day of infection. The significant increase in cytokines on the5th day of infection in the Phosphorus group is related to a significant decrease in the number of inflammatory foci in cardiac tissue on the 10th day of infection in this group. DISCUSSION AND CONCLUSION: Treatment with Phosphorus 13cH promotes beneficial effects in T. cruzi infection in Wistar rats by modulating the secretion of IFN-γ and TNF-α with decreased inflammation in cardiac tissue. These results reinforce the importance of considering the use of homeopathy for establishing new therapeutic approaches in the management of patients with Chagas disease.

Increased of the hepatocytes and splenocytes apoptosis accompanies clinical improvement and higher survival in mice infected with Trypanosoma cruzi and treated with highly diluted Lycopodium clavatum.

Recent evidence includes apoptosis as a defense against Trypanosoma cruzi infection, which promotes an immune response in the host induced by T cells, type 1, 2 and 17. Currently, there is no medicine completely preventing the progression of this disease. We investigated the immunological and apoptotic effects, morbidity and survival of mice infected with T. cruzi and treated with dynamized homeopathic compounds 13c: Kalium causticum (GCaus), Conium maculatum, (GCon), Lycopodium clavatum (GLy) and 7% alcohol solution (control, vehicle compounds, GCI). There was significant difference in the increase of apoptosis in the treated groups, compared with GCI, which might indicate action of the compounds in these cells. Infected animals treated with Lycopodium clavatum presented better performance compared with other groups. GLy showed a higher amount of hepatocytes and splenocytes undergoing apoptosis, higher number of apoptotic bodies in the liver, predominance of Th1 response, increased TNF-α and decreased IL-6, higher survival, lower morbidity, higher water consumption, body temperature, tendency to higher feed intake and weight gain compared with GCI. Conium maculatum had worse results with increased Th2 response with increased IL-4, worsening of the infection with early mortality of the animals. Together, these data suggest that highly diluted medicines modulate the immune response and apoptosis, affecting the morbidity of animals infected with a highly virulent strain of T. cruzi, being able to minimize the course of infection, providing more alternative approaches in the treatment of Chagas disease.

Beneficial immunomodulatory and neuro digestive effect in Trypanosoma cruzi infection after Lycopodium clavatum 13c treatment.

Studies show that highly diluted medications demonstrate benefits in treating infections, constituting an alternative for their treatment. The present study evaluated the effects of Lycopodium clavatum, dynamization 13c, in Wistar rats infected with T. cruzi. In this study 42 male rats were intraperitoneally inoculated with T. cruzi - Y strain and allocated into groups: IC (infected control group) and Ly (treated with L. clavatum 13c). The cytokines dosage (IFN-γ, IL-12, IL-10, IL-4), quantification and morphometry of myenteric neurons were evaluated. The treatment with L. clavatum modifies the immune response, with increase of IFN-γ on day 10 a.i. and IL-12 on day 24 a.i., decrease of IL-10 concentration on day 10 a.i. and subsequent increase of this cytokine and IL-4 on day 24 a.i., affording a bigger number of myenteric neurons compared to IC group. Thus, L. clavatum 13c promoted on rats infected with T. cruzi a beneficial immunomodulatory action reducing the pathogenic progression of digestive Chagas disease.

Different treatment schemes and dynamizations of Trypanosoma cruzi biotherapies: what information do they transfer to the organism in infected mice?

BACKGROUND: The use of biotherapies in Trypanosoma cruzi infection can provide an understanding about effects of these highly diluted medications. OBJECTIVES: To evaluate different treatment schemes and dynamizations of biotherapies prepared from blood trypomastigotes (buffy coat) in mice infected with T. cruzi. METHODS: Swiss mice infected with Y strain of T. cruzi were divided into two experiments. Experiment 1, all treated groups received biotherapy 7dH (10 µL/mL ad libitum) in different treatment schemes: TB7dH - treated 3 days before infection; TBA7dH - treated 3 days before and after infection; TBAe.d.7dH - treated 3 days before infection and every day after infection and IC - infection control. Experiment 2, all treated groups received medication in different dynamizations 3 days before and after infection (10 µL/mL ad libitum): TBA15dH - treated with biotherapy 15dH; TBA16dH - treated with biotherapy 16dH; TBA17dH - treated with biotherapy 17dH; TBAp.chords - treated with biotherapy 'potency chords' and IC - infection control. We evaluated parasitological and clinical parameters. RESULTS: Experiment 1 showed that different treatment schemes with biotherapy 7dH produced different effects on infection evolution. TBA7dH group had the best outcome, with lower parasitemia, higher survival, and better clinical evolution compared to IC. Experiment 2 showed that biotherapy 'potency chords' had effects different from the individual dynamizations that it contained (15dH, 16dH, and 17dH). Animals that had patent parasitemia had delayed emergence of parasites in blood and subsequent increase in parasitemia, but had better clinical evolution compared to IC. CONCLUSIONS: The effects of T. cruzi biotherapies depend on frequency at which they are administered, dynamization, and host-parasite relationship/individual susceptibility of treated organism. Biotherapy appeared to transfer to infected organism 'antigenic information' related to parasite and 'disease information' related to molecules produced by host's immune response and contained in the buffy coat used to prepare the medication.

Highly diluted medication reduces tissue parasitism and inflammation in mice infected by Trypanosoma cruzi.

AIM: To evaluate the effects of Kalium causticum, Conium maculatum, and Lycopodium clavatum 13cH in mice infected by Trypanosoma cruzi. MATERIALS AND METHODS: In a blind, controlled, randomized study, 102 male Swiss mice, 8 weeks old, were inoculated with 1400 trypomastigotes of the Y strain of T. cruzi and distributed into the following groups: CI (treated with 7% hydroalcoholic solution), Ca (treated with Kalium causticum 13cH), Co (treated with Conium maculatum 13cH), and Ly (treated with Lycopodium clavatum 13cH). The treatments were performed 48 h before and 48, 96, and 144 h after infection. The medication was repertorized and prepared in 13cH, according to Brazilian Homeopathic Pharmacopoeia. The following parameters were evaluated: infectivity, prepatent period, parasitemia peak, total parasitemia, tissue tropism, inflammatory infiltrate, and survival. Statistical analysis was conduced considering 5% of significance. RESULTS: The prepatent period was greater in the Ly group than in the CI group (p = 0.02). The number of trypomastigotes on the 8th day after infection was lower in the Ca group than in the CI group (p < 0.05). Total parasitemia was significantly lower in the Ca, Co, and Ly groups than in the CI group. On the 12th day after infection, the Ca, Co, and Ly groups had fewer nests and amastigotes/nest in the heart than the CI group (p < 0.05). Decreases in the number of nests and amastigotes in the intestine were observed in the Ly group compared with the CI group (p < 0.05). In the liver (day 12), Ly significantly prevented the formation of inflammatory foci compared with the other groups. In skeletal muscle, Co and Ly decreased the formation of inflammatory foci compared with CI (p < 0.05). Ly afforded greater animal survival compared with CI, Ca, and Co (p < 0.05). The animals in the Co group died prematurely compared with the CI group (p = 0.03). CONCLUSIONS: Ly with 13cH potency had significantly more benefits in the treatment of mice infected with T. cruzi, reducing the number of blood parasites, amastigote nests in tissue, and the number of amastigotes per nest and increasing animal survival.

Different forms of administration of biotherapy 7dH in mice experimentally infected by Trypanosoma cruzi produce different effects.

OBJECTIVE: To evaluate the effects of different forms of administration of the blood trypomastigotes biotherapy 7dH in mice experimentally infected with Trypanosoma cruzi. MATERIAL AND METHODS: Male swiss mice were inoculated with 1400 blood trypomastigotes of the Y strain of T. cruzi and allocated into 5 treatment groups: IC (distilled water); TCBZ (benznidazole); TBA(7dH) (biotherapy 7dH 20 days after infection); TBB(7dH)7 (biotherapy 7dH seven days before infection); TBB(7dH)30 (biotherapy 7dH 30 days before infection). Parasitological parameters assessed included pre-patent and patent periods, parasitemia peak, total parasitemia, mortality and survival rates. Cure index was obtained by fresh blood examination, hemoculture and polymerase chain reaction (PCR). RESULTS: The TBB(7dH)7 group showed a reduction in parasitemia peak, parasitemia area under the curve and total parasitemia. TBB(7dH)30 showed a tendency to increased pre-patent and survival periods, peak parasitemia was increased without increased total parasitemia. TBA(7dH) did not present significant alterations in the parasitological parameters analyzed. CONCLUSIONS: Biotherapy 7dH given before infection (7 or 30 days) produces different effects suggesting modulation of the host's immune system. The effects range from reduced parasitemia to its effective increase. The use of biotherapy to treat T. cruzi infection including dose, potency and schedule deserves further investigation.

Canova medication modifies parasitological parameters in mice infected with Trypanosoma cruzi.

The goals of this study were to evaluate the effect of the Canova medication, a homeopathic immune-system modulator, on the evolution of infection induced by the Trypanosoma cruzi Y strain in mice. The animals were divided into five groups: (i) untreated infected controls (I), (ii) infected animals treated with benznidazole (Bz), (iii) infected animals treated with the Canova medication (CM), (iv) infected animals treated with benznidazole and the Canova medication (Bz+CM), and (v) uninfected controls that received only the vehicle (grain alcohol) (C). The parameters evaluated were: parasitemia, mortality, control of cure, and tissue parasitism analysis. Our results showed that the evolution of the experimental infection was modified by treatment with CM, and that daily and consecutive doses were harmful to the animals, causing death in 100% of the infected animals in a brief period. The analysis of parasitism performed on the organs on the 12th day postinfection showed that in infected animals treated with CM, the number of amastigote/nests in the spleen was significantly reduced, while in cardiac tissue, intestine, and liver the number was significantly increased compared with infected control animals. These results indicate that CM has a negative influence on the host-parasite relationship, modifying the tropism of the parasite for tissues, and increasing the parasitemia peak in this experimental model.

Effects of Highly Diluted Drugs on Experimental Infection with Trypanosoma cruzi In Vivo: Systematic Review.

Objective: To investigate, through a systematic review, the effects of the use of highly diluted drugs in the treatment of experimental infection with Trypanosoma cruzi. Design: The authors searched for scientific publications in the databases PubMed, Web of Science, SCOPUS, LILACS, and the Google Scholar search system, from 2000 to 2018, following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement. According to the criteria established, a total of 22 studies were included. Settings/Location: The study took place at the State University of Maringá, Maringá, PR, Brazil. Subjects: Male mice (Mus musculus) or rats (Rattus norvegicus). Interventions: Highly diluted drugs. Outcome measures: The parameters evaluated in the studies were parasitological, clinical, immunological, histopathological and hematological. Results: The studies demonstrated that the effects of highly diluted drugs are related to their dynamizations, treatment regimen, and host susceptibility to T. cruzi infection, and depend on the initial information transmitted to the treated organism, making this information the "model" of how the treated organism will react. Regardless of the mechanism of action, these drugs provide a decrease in inflammation, which is one of the central phenomena of the pathogenesis of T. cruzi infection. Conclusions: This systematic review brings out the importance of the T. cruzi infection model as a reliable and valid model for studying different effects produced by highly diluted drugs. Considering the findings and in a broader perspective, this study contributes to considering these drugs as a possible way of dealing with "treatment" in general, presents the need to reexamine the biochemical model and develop a model for the effect of high dilutions in general, as well as for the treatment of parasitic infections.

Changes of RAPD profile of Trypanosoma cruzi II with Canova and Benznidazole.

Chagas disease, caused by the protozoan Trypanosoma cruzi, involves immunomediated processes. Canova (CA) is a homeopathic treatment indicated in the diseases in which the immune system is depressed. This study evaluated the Random Amplification of Polymorphic DNA (RAPD) profile of T. cruzi under the influence of CA and Benznidazole (BZ). Mice infected with the genetic lineage of T. cruzi II (Y strain) were divided into 4 groups: Infected animals treated with saline solution (control group); treated with CA; treated with BZ; treated with CA and BZ combined. Treatment was given at the 5th-25th days of infection (D5-25). The parasites were isolated by haemoculture in Liver Infusion Tryptose (LIT) medium: at D5 (before treatment), D13, 15 and 25 (during treatment) and D55 and 295 (after treatment). DNA was extracted from the mass of parasites. RAPD was done with the primers lambdagt11-F, M13F-40 and L15996, the amplified products were eletrophoresed through a 4% polyacrylamide gel. Data were analyzed by the coefficient of similarity using the DNA-POP program. 163 markers were identified, 5 of them monomorphic. CA did not act against the parasites when used alone. The RAPD profiles of parasites treated with BZ and CA+BZ were different from those in the control group and in the group treated with CA. The actions of the CA and BZ were different and the action of BZ was different from the action of CA+BZ. These data suggest that CA may interact with BZ. The differences in the RAPD profile of the Y strain of T. cruzi produced by BZ, CA+BZ and the natural course of the infection suggest selection/suppression of populations.

Effects of homeopathy in mice experimentally infected with Trypanosoma cruzi.

AIM: The aim of this study was to evaluate the action of homeopathic treatment on mice experimentally infected with Trypanosoma cruzi. METHODS: Eighty adult male C57BL/6 inbred mice were randomly allocated to five groups treated with biotherapy (nosode) of T. cruzi 12dH (12x) pre- and post-infection; Phosphorus 12dH post-infection; infected control treated with control solution and uninfected control. The biotherapy was prepared by the Costa method from the blood of mice experimentally infected with the Y strain of T. cruzi. Phosphorus was used because of its clinical and reportorial similarity to Chagas disease. T. cruzi (10(4)) sanguineous forms were inoculated intraperitoneally per animal. Parasitaemia was monitored, leukocyte and serological responses were evaluated at 0, 7, 14 and 42 days after infection. The prepatent and patent periods of parasitaemia, maximum of parasitaemia, day of maximum parasitaemia and mortality rates were compared between groups. RESULTS: A significantly shorter period of patent parasitaemia was observed in the group treated with the biotherapy before infection (p<0.05) than in the other groups. This group also had the lowest parasitaemias values at 9, 13, 15 (p<0.05), 17 (p<0.05), 22, 24 and 28 days, a lower rate of mortality and a significant increase of lymphocytes compared to the infected control group. The Phosphorus group had the longest period of patent parasitaemia, higher maximum parasitaemia, and a significant reduction of lymphocyte numbers, but no mortality. The infected control group had the highest mortality rate (not statistically significant), and the highest IgG titres at 42 days post-infection (p<0.05). CONCLUSIONS: The results suggest that pre-treatment with biotherapy modulates host immune response to T. cruzi, mainly during the acute phase of the infection. Phosphorus shows an action on the pathogenicity by T. cruzi infection. Homeopathic treatment of T. cruzi infection should be further investigated.

Current drug therapy and pharmaceutical challenges for Chagas disease.

One of the most significant health problems in the American continent in terms of human health, and socioeconomic impact is Chagas disease, caused by the protozoan parasite Trypanosoma cruzi. Infection was originally transmitted by reduviid insects, congenitally from mother to fetus, and by oral ingestion in sylvatic/rural environments, but blood transfusions, organ transplants, laboratory accidents, and sharing of contaminated syringes also contribute to modern day transmission. Likewise, Chagas disease used to be endemic from Northern Mexico to Argentina, but migrations have earned it global. The parasite has a complex life cycle, infecting different species, and invading a variety of cells - including muscle and nerve cells of the heart and gastrointestinal tract - in the mammalian host. Human infection outcome is a potentially fatal cardiomyopathy, and gastrointestinal tract lesions. In absence of a vaccine, vector control and treatment of patients are the only tools to control the disease. Unfortunately, the only drugs now available for Chagas' disease, Nifurtimox and Benznidazole, are relatively toxic for adult patients, and require prolonged administration. Benznidazole is the first choice for Chagas disease treatment due to its lower side effects than Nifurtimox. However, different strategies are being sought to overcome Benznidazole's toxicity including shorter or intermittent administration schedules-either alone or in combination with other drugs. In addition, a long list of compounds has shown trypanocidal activity, ranging from natural products to specially designed molecules, re-purposing drugs commercialized to treat other maladies, and homeopathy. In the present review, we will briefly summarize the upturns of current treatment of Chagas disease, discuss the increment on research and scientific publications about this topic, and give an overview of the state-of-the-art research aiming to produce an alternative medication to treat T. cruzi infection.

Highly diluted medication reduces parasitemia and improves experimental infection evolution by Trypanosoma cruzi.

BACKGROUND: There is no published information about the use of different protocols to administer a highly diluted medication.Evaluate the effect of different protocols for treatment with biotherapic T. cruzi 17 dH (BIOT(Tc17dH)) on clinical/parasitological evolution of mice infected with T. cruzi-Y strain. METHODS: A blind, randomized controlled trial was performed twice, using 60 28-day-old male Swiss mice infected with T. cruzi-Y strain, in five treatment groups: CI - treated with a 7% ethanol-water solution, diluted in water (10 µL/mL) ad libitum; BIOT(PI) - treated with BIOT(Tc17dH) in water (10 µL/mL) ad libitum during a period that started on the day of infection; BIOT(4DI) - treated with BIOT(Tc17dH) in water (10 µL/mL) ad libitum beginning on the 4th day of infection; BIOT(4-5-6) - treated with BIOT(Tc17dH) by gavage (0.2 mL/ animal/day) on the 4th, 5th and 6th days after infection; BIOT(7-8-9) - treated with BIOT(Tc17dH) by gavage (0.2 mL/ animal/day) on the 7th, 8th and 9th days after infection. We evaluated: parasitemia; total parasitemia (P(total)); maximum peak of parasites; prepatent period (PPP) - time from infection to detection of the parasite in blood; patent period (PP) - period when the parasitemia can be detected in blood; clinical aspects; and mortality. RESULTS: Parasitological parameters in the BIOT(PI) and mainly in the BIOT(4PI) group showed better evolution of the infection compared to the control group (CI), with lower P(total), lower maximum peak of parasites, higher PPP, lower PP and longer survival times. These animals showed stable body temperature and higher weight gain and water consumption, with more animals having normal-appearing fur for longer periods. In contrast, groups BIOT(4-5-6) and BIOT(7-8-9) showed worse evolution of the infection compared to the control group, considering both parasitological and clinical parameters. The correlation analysis combined with the other data from this study indicated that the prepatent period is the best parameter to evaluate the effect of a medication in this model. CONCLUSIONS: The BIOT(4DI) group showed the best clinical and parasitological evolution, with lower parasitemia and a trend toward lower mortality and a longer survival period. The prepatent period was the best parameter to evaluate the effect of a medication in this model.

Canova, a Brazilian medical formulation, alters oxidative metabolism of mice macrophages.

Macrophages play a significant role in the host defence mechanism. When activated they can produce reactive oxygen species (ROS) as well as related reactive nitrogen species (RNS). ROS are produced via NAD(P)H oxidase which catalyzes superoxide (O2-) formation. It is subsequently converted to hydrogen peroxide (H2O2) by either spontaneous or enzyme-mediated dismutation. Nitric oxide synthase (NOS) catalyzes nitric oxide (NO) formation. Canova (CA) is a Brazilian medication produced with homeopathic techniques, composed of Aconitum, Thuya, Bryonia, Arsenicum, Lachesis in distilled water containing less than 1% ethanol. Previous studies demonstrated that CA is neither toxic nor mutagenic and activates macrophages decreasing the tumor necrosis factor-alpha (TNFalpha) production. In this assay we showed that macrophages triggered with Canova increased NAD(P)H oxidase activity as well as that of iNOS, consequently producing ROS and NO respectively. Cytochrome oxidase and peroxisomes activities were inhibited by NO. As NO and O2- are being produced at the same time, formation of peroxynitrite (ONOO-) may be occurring. A potential explanation is provided on how treatment with Canova may enhance immune functions which could be particularly important in the cytotoxic actions of macrophages. CA can be considered as a new adjuvant therapeutic approach to known therapies.

Estudo comparativo dos indices de cura de camundongos tratados com quimioterapicos e trypanosominum (TC D 30) / Comparative analysis of the cure rate in mices treated with chemoterapics and trypanosominum (TC D 30)

Trypanosoma cruzi strain isolated from wild mamals or from man and studied in the laboratory show different characteristics that permit their individualization. Blood trypamastigotes exhibit different morphological patterns according to the strain studied. Tropism for different tissues has also been reported. Morphologically, there are wide, thin and intermediate forms wose proportions vary. The results obtained by different investigations in the treatment of Chagas disease are discrepant in different geographic areas, probably because of a relationship with different populations of T.cruzi. In the presentestudy, we investigated experimentally the susceptibility of two polar species, i.e. two species with predominance of thin and wide forms, respectively (Y e Bolivia strains) to the action of Nifurtimax, Benzonidazol and Trypanosaminum (TC D30). Groups of 30 mice aged 60 days were inoculated with 10.4 bloodstream forms of the Y and Bolivia strains of T.cruzi. On the third day of infection, the animals were treated with 100 mg/Kg of both chemotherapeutic agents and with 5 dropsof Trypanosaminum (TC D 30) orally for 20 days. Each animal was evaluated for parasitemia and behavior. Xenodiagnosis and blood cultures were carried out after 40 days. The results showed: a) a sharp resistance of the Bolivia strain to the chemotherapeutic agents, while the Y strain was more susceptible; b) identical susceptibility of the Y and Bolivia strains to treatment with Trypanosominum. We infer from these data that the chemotherapeutic agents act on the parasitic formsby destroying them or not, with sharp differences when different populations areinvolved. In contrast, Trypanosaminum (TC D 30) may act on the stimulating the imunologic response, wich may destroy the etiologic agents through a specific response mechanism, regardless of the characteristics of the strain studied