Rattus norvegicus infectados com Trypanosoma cruzi: efeitos clínicos do tratamento com Lycopodium clavatun e phosphorus ultradiluídos / Rattus norvegicus infected with Trypanosoma cruzi: clinical effects of treatment with highly diluted Lycopodium clavatun and phosphorus / Rattus norvegicus infectado con Trypanosoma cruzi: efectos clínicos del tratamiento con Clavatun licopodio altamente diluido y fósforo

A doença de Chagas causada pelo parasita Trypanosoma cruzi acomete milhões de pessoas no mundo e não conta com um medicamento de ação efetiva para o seu tratamento etiológico. As drogas disponíveis, o nifurtimox e o benznidazol possuem índices de cura baixos com efeitos colaterais e toxidade que dificultam a adesão dos pacientes à terapia. Este fato impulsiona a busca por alternativas de tratamento que sejam mais efetivas e menos agressivas. Sendo assim, este trabalho teve como objetivo a avaliação dos efeitos clínicos apresentados por Rattus norvergicus infectados por T. cruzi e tratados com soluções ultradiluídas de Lycopodium clavatum ou Phosphorus. O estudo envolveu 93 ratos com quarenta e cinco dias de idade infectados intraperitonealmente com 5x106 formas tripomastigotas sanguíneos da cepa Y de T. cruzi, distribuídos nos grupos: Sadio SD (n=13) - controle não infectado e não tratado, grupo CI (n=27) - controle infectado e tratado com solução hidroalccólica 7% (etanol ­ água), grupo LY diluição 1:1x1026 (n=27) - infectado e tratado com Lycopodium, grupo PH diluição 1:1x1026 (n=26) - infectado e tratado com Phosphorum. Os animais foram avaliados clinicamente através dos parâmetros peso, temperatura, consumo de água e ração, quantidade de excretas, diâmetro e comprimento intestinal, aspecto da pelagem e consistência das fezes. Este estudo mostrou que os parâmetros utilizados foram importantes para a definição clínica da infecção de Rattus novergicus, linhagem Wistar pelo T. cruzi. Mostrou que os medicamentos LY e PH apresentam efeitos benéficos na evolução da clínica dos animais tratados. A utilização de Lycopodium clavatum e Phosphorus diluídos na proporção de 1:1x1026, apresentaram efeitos diferentes. Oito e seis parâmetros de quatorze analisados mostraram efeitos positivos para LY e PH, respectivamente. Os parâmetros consumo de água e ração, quantidade de excretas, diarreia, alopecia difusa e comprimento intestinal apresentaram diferenças significativas em relação ao controle infectado mostrando que mais estudos são necessários com o uso de medicamentos ultradiluídos na infecção pelo T. cruzi.

Chagas disease caused by the parasite Trypanosoma cruzi affects millions of people worldwide and does not have an effective drug for its etiological treatment. The available drugs, nifurtimox and benznidazole, have low cure rates with side effects and toxicity that make it difficult for patients to adhere to therapy. This fact drives the search for treatment alternatives that are more effective and less aggressive. Therefore, this work aimed to evaluate the clinical effects presented by Rattus norvergicus infected by T. cruzi and treated with ultradiluted solutions of Lycopodium clavatum or Phosphorus. The study involved 93 forty five day old rats intraperitoneally infected with 5x106 blood trypomastigotes forms of the Y strain of T. cruzi, distributed in the following groups: Healthy SD (n=13) - non-infected and untreated control, CI group (n =27) - infected control and treated with 7% hydroalcoholic solution (ethanol ­ water), LY group dilution 1:1x1026 (n=27) - infected and treated with Lycopodium, PH group dilution 1:1x1026 (n=26) - infected and treated with Phosphorum. The animals were clinically evaluated through the parameters weight, temperature, water and feed consumption, amount of excreta, intestinal diameter and length, coat appearance and feces consistency. This study showed that the parameters used were important for the clinical definition of infection of Rattus novergicus, Wistar lineage by T. cruzi. It showed that LY and PH drugs have beneficial effects on the clinical evolution of treated animals. The use of Lycopodium clavatum and Phosphorus diluted in the ratio of 1:1x1026, showed different effects. Eight and six parameters out of fourteen analyzed showed positive effects for LY and PH, respectively. The parameters water and feed consumption, amount of excreta, diarrhea, diffuse alopecia and intestinal length showed significant differences in relation to the infected control, showing that more studies are needed with the use of ultradiluted drugs in T. cruzi infection.

La enfermedad de Chagas causada por el parásito Trypanosoma cruzi afecta a millones de personas en todo el mundo y no cuenta con un fármaco eficaz para su tratamiento etiológico. Los fármacos disponibles, nifurtimox y benznidazol, presentan bajas tasas de curación con efectos secundarios y toxicidad que dificultan la adherencia terapéutica de los pacientes. Este hecho impulsa la búsqueda de alternativas de tratamiento más eficaces y menos agresivas. Por lo tanto, este trabajo tuvo como objetivo evaluar los efectos clínicos presentados por Rattus norvergicus infectados por T. cruzi y tratados con soluciones ultradiluidas de Lycopodium clavatum o Fósforo. En el estudio participaron 93 ratas de cuarenta y cinco días de edad infectadas intraperitonealmente con 5x106 formas tripomastigotes sanguíneas de la cepa Y de T. cruzi, distribuidos en los siguientes grupos: SD sano (n=13) - control no infectado y no tratado, grupo CI (n =27) - control infectado y tratado con solución hidroalcohólica al 7% (etanol - agua), grupo LY dilución 1:1x1026 (n=27) - infectado y tratado con Lycopodium, grupo PH dilución 1:1x1026 (n=26) - infectado y tratado con Phosphorum. Los animales fueron evaluados clínicamente mediante los parámetros peso, temperatura, consumo de agua y pienso, cantidad de excrementos, diámetro y longitud intestinal, aspecto del pelaje y consistencia de las heces. Este estudio demostró que los parámetros utilizados eran importantes para la definición clínica de la infección de Rattus novergicus, linaje Wistar por T. cruzi. Demostró que los fármacos LY y PH tienen efectos beneficiosos en la evolución clínica de los animales tratados. El uso de Lycopodium clavatum y Phosphorus diluidos en la proporción de 1:1x1026, mostró efectos diferentes. Ocho y seis parámetros de los catorce analizados mostraron efectos positivos para LY y PH, respectivamente. Los parámetros consumo de agua y pienso, cantidad de excretas, diarrea, alopecia difusa y longitud intestinal mostraron diferencias significativas en relación al control infectado, mostrando que son necesarios más estudios con el uso de fármacos ultradiluidos en la infección por

Experimental infection of Rhodnius robustus Larrousse, 1927 (Hemiptera, Reduviidae, Triatominae) with Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae) IV.

Vector competence of triatomines (kissing bugs) for Trypanosoma cruzi transmission depends on the parasite-vector interaction and the genetic constitution of both. This study evaluates the susceptibility and vector competence of Rhodnius robustus experimentally infected with T. cruzi IV (TcIV). Nymphs were fed on infected mice or an artificial feeder with blood containing culture-derived metacyclic trypomastigotes (CMT) or blood trypomastigotes (BT). The intestinal contents (IC) and excreta of the insects were examined by fresh examination and kDNA-PCR. The rate of metacyclogenesis was also determined by differential counts. Fifth instar nymphs fed with CMT ingested a greater blood volume (mean of 74.5 µL) and a greater amount of parasites (mean of 149,000 CMT/µL), and had higher positivity in the fresh examination of the IC. Third instar nymphs fed with CMT had higher positivity (33.3%) in the fresh examination of the excreta. On the 20th day after infection (dai), infective metacyclic trypomastigote (MT) forms were predominant in the excreta of 3/4 experimental groups, and on the 30th dai, the different parasitic forms were observed in the IC of all the groups. Higher percentages of MT were observed in the excreta of the 5th instar nymphs group (84.1%) and in the IC of the 3rd instar nymphs group (80.0%). Rhodnius robustus presented high susceptibility to infection since all nymphs were infected, regardless of the method used for blood meal, in addition these insects demonstrated vector competence for TcIV with high rates of metacyclogenesis being evident.

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.

Treatment with Lycopodium clavatum 200dH Intensifies Kidney and Liver Injury in Mice Infected with Toxoplasma gondii.

The effects of infection with Toxoplasma gondii vary from asymptomatic to the development of alterations in various organs (including the liver and kidneys) which may be irreversible, and lead to the death of the host. Whereas homeopathy is an alternative and effective method for treating various diseases, including those caused by protozoa, we questioned the effect of using Lycopodium clavatum in mice infected with T. gondii. One hundred male Swiss mice, 60 days old, were divided into four groups (n = 25/group): NIC (uninfected and untreated control), IC (infected and treated with un-dynamized 7% alcohol solution [vehicle]), G48 (infected and treated 48 h before infection and treated three more times; at 2, 4, and 6 days post-infection (dpi) with L. clavatum 200dH), and G72 (infected and treated for 3 consecutive days before infection with L. clavatum 200dH). In this study, physiological, histopathological, and immunological parameters were evaluated. The L. clavatum 200dH intensified renal damage in mice infected with T. gondii from 7 dpi, causing severe and progressive alterations during this period, such as various degrees of inflammation, edema, atrophy, and tubular cystic dilation, degenerated tubules with intra-cytoplasmic vacuoles and coalescing spots, severe vascular lesions, glomerulonephritis, and peri-glomerular congestion. In the G72 animals, which received L. clavatum 200dH, more severe cortex damage was observed (91.66-96.66%) as compared to the IC group (55-80%) and more renal corpuscle, and renal tubule injury was observed (80 ± 5 to 96.7% ± 2.89 of the total area) during all periods, as compared to the IC group (p < 0.05). Both groups presented high liver enzyme levels, and the highest values for AST were observable at 60 dpi. We observed significant increases of type I and III collagen, as well as high levels of TGF-ß1 in both organs of the treated animals, the main factor involved in fibrosis in areas damaged by the process. L. clavatum 200dH intensifies kidney and liver alterations in mice infected with T. gondii. Our results reinforce caution when indicating administration schemes and dosages for ultra-diluted drugs.

Analyzing weight evolution in mice infected by Trypanosoma cruzi

Concerning the specificities of a longitudinal study, the trajectories of a subject's mean responses not always present a linear behavior, which calls for tools that take into account the non-linearity of individual trajectories and that describe them towards associating possible random effects with each individual. Generalized additive mixed models (GAMMs) have come to solve this problem, since, in this class of models, it is possible to assign specific random effects to individuals, in addition to rewriting the linear term by summing unknown smooth functions, not parametrically specified, then using the P-splines smoothing technique. Thus, this article aims to introduce this methodology applied to a dataset referring to an experiment involving 57 Swiss mice infected by Trypanosoma cruzi, which had their weights monitored for 12 weeks. The analyses showed significant differences in the weight trajectory of the individuals by treatment group; besides, the assumptions required to validate the model were met. Therefore, it is possible to conclude that this methodology is satisfactory in modeling data of longitudinal sort, because, with this approach, in addition to the possibility of including fixed and random effects, these models allow adding complex correlation structures to residuals.

Linear mixed model for weight analysis in mice infected by Trypanosoma cruzi

The use of linear mixed models for nested structure longitudinal data is called hierarchical linear modeling. Thismodeling takes into account the dependence of existing data within each level and between hierarchical levels. The process of modeling, estimating and analyzing diagnoses was illustrated through data on the weights of mice experimentally infected by Trypanosoma cruzi, divided into different treatment groups, with the purpose of verifying the evolution of their body weight as a result of usingdifferent types of biotherapeutics produced from Gallus gallus domesticus(chicken) serum to treat Trypanosoma cruzi. Through the model selection criteria AIC and BIC and the likelihood ratio test, a model was chosen to describe the data correctly. Model diagnoses were then performed by means of residual analysis for both levels and an analysis of influential observations to verify if any observations were signaled as influencing the fixed effects, the components of variance and the adjusted values. After the analysis, it was possible to notice that the observations that were signaled as influential had little impact on the Model chosen initially, so it was maintained, with no differences being evidenced between the treatments with the biotherapeutics tested; only the Time variable and the Random intercept were necessary to describe the weight of the mice.

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.

Sympatry influence in the interaction of Trypanosoma cruzi with triatomine.

INTRODUCTION: Trypanosoma cruzi, the etiologic agent of Chagas disease, is widely distributed in nature, circulating between triatomine bugs and sylvatic mammals, and has large genetic diversity. Both the vector species and the genetic lineages of T. cruzi present a varied geographical distribution. This study aimed to verify the influence of sympatry in the interaction of T. cruzi with triatomines. Methods: The behavior of the strains PR2256 (T. cruzi II) and AM14 (T. cruzi IV) was studied in Triatoma sordida (TS) and Rhodnius robustus (RR). Eleven fifth-stage nymphs were fed by artificial xenodiagnosis with 5.6 × 103 blood trypomastigotes/0.1mL of each T. cruzi strain. Every 20 days, their excreta were examined for up to 100 days, and every 30 days, the intestinal content was examined for up to 120 days, by parasitological (fresh examination and differential count with Giemsa-stained smears) and molecular (PCR) methods. Rates of infectivity, metacyclogenesis and mortality, and mean number of parasites per insect and of excreted parasites were determined. RESULTS: Sympatric groups RR+AM14 and TS+PR2256 showed higher values of the four parameters, except for mortality rate, which was higher (27.3%) in the TS+AM14 group. General infectivity was 72.7%, which was mainly proven by PCR, showing the following decreasing order: RR+AM14 (100%), TS+PR2256 (81.8%), RR+PR2256 (72.7%) and TS+AM14 (36.4%). CONCLUSIONS: Our working hypothesis was confirmed once higher infectivity and vector capacity (flagellate production and elimination of infective metacyclic forms) were recorded in the groups that contained sympatric T. cruzi lineages and triatomine species.

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.

Sympatry influence in the interaction of Trypanosoma cruzi with triatomine

Abstract INTRODUCTION: Trypanosoma cruzi, the etiologic agent of Chagas disease, is widely distributed in nature, circulating between triatomine bugs and sylvatic mammals, and has large genetic diversity. Both the vector species and the genetic lineages of T. cruzi present a varied geographical distribution. This study aimed to verify the influence of sympatry in the interaction of T. cruzi with triatomines. Methods: The behavior of the strains PR2256 (T. cruzi II) and AM14 (T. cruzi IV) was studied in Triatoma sordida (TS) and Rhodnius robustus (RR). Eleven fifth-stage nymphs were fed by artificial xenodiagnosis with 5.6 × 103 blood trypomastigotes/0.1mL of each T. cruzi strain. Every 20 days, their excreta were examined for up to 100 days, and every 30 days, the intestinal content was examined for up to 120 days, by parasitological (fresh examination and differential count with Giemsa-stained smears) and molecular (PCR) methods. Rates of infectivity, metacyclogenesis and mortality, and mean number of parasites per insect and of excreted parasites were determined. RESULTS: Sympatric groups RR+AM14 and TS+PR2256 showed higher values of the four parameters, except for mortality rate, which was higher (27.3%) in the TS+AM14 group. General infectivity was 72.7%, which was mainly proven by PCR, showing the following decreasing order: RR+AM14 (100%), TS+PR2256 (81.8%), RR+PR2256 (72.7%) and TS+AM14 (36.4%). CONCLUSIONS: Our working hypothesis was confirmed once higher infectivity and vector capacity (flagellate production and elimination of infective metacyclic forms) were recorded in the groups that contained sympatric T. cruzi lineages and triatomine species.

Dynamized ethyl alcohol improves immune response and behavior in murine infection with Trypanosoma cruzi.

AIM: To evaluate the effects of dynamized ethyl alcohol (Ethylicum)6cH and 30cH in mice infected with T. cruzi. METHODS: In a blind, randomized and controlled assay, 63 eight-week-old, Swiss, male mice, infected with IP (1400 trypomastigotes, T. cruzi-Y-strain), were allocated into groups: CNI-non-infected (n=12), CI-infected and non-treated (n=17), Et6cH-infected, treated with Ethylicum 6cH (dilution 1:1012) (n=17), Et30cH-infected, treated with Ethylicum 30cH (dilution 1:1060) (n=17). Treatment was administered 48h before and after infection, followed by 56h/56h periods, until the 9th day after infection (a.i), for 16 h. Survival and mortality were assessed until the 82nd day after infection (a.i.). TNF-α, IL-6, IL-10, IL-5 and IL-17A cytokines were assessed in serum (3-4 animals/group), at time T0 (before infection), T8 and T12 (8th and 12th a.i), using the Mouse Cytokine 20-Plex Panel Magnetic Kit (Invitrogen, USA). Inflammation was determined in heart sections (eosin-hematoxylin staining) and behavior was analyzed with ANY-maze® software. The study was approved by the Animal Ethics Committee/UEM. Statistica 8.0 and R 3.0.2 software were used for statistical analyses. RESULTS: The greater survival observed in the Et6cH group was related to decreased inflammation in heart tissue and increased IL-5 at T0 (p<0.05) and IL-10 at T8 (p<0.05), characterizing the Th2 response. It was also related to shorter periods of immobility, observed on day 12 a.i. The higher mortality in the Et30cH group was related to increased inflammation in the heart and a higher concentration of IL-6 and TNF-α cytokines, characterizing the Th1 response. CONCLUSION: The results demonstrate the beneficial effect of Ethylicum 6cH in acute murine infection by T. cruzi.

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.

Homeopathic medicines cause Th1 predominance and induce spleen and megakaryocytes changes in BALB/c mice infected with Leishmania infantum.

The prevalence of Th1/Th2 response, spleen changes and megakaryocytes were investigated in BALB/c mice (n=138) infected with Leishmania infantum, and treated with Leishmania infantum 30× (10-30) biotherapy - BioLi30×. We performed controlled experiments using 8-to-12-week-old mice, infected with 5×107L. infantum promastigotes, divided into eight groups: G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30× pre-treated), G4 (BioLi30× pre/post-treated), G5 (BioLi30× post-treated), G6 (Water 30× post-treated), G7 (Antimonium crudum 30× post-treated) and G8 (Glucantime® post-treated). G3-G7 groups were orally treated with their respective drugs diluted in filtered water (1:10), and G8 received Glucantime® (0.6mg/100µl of PBS), intraperitoneally. Spleen fragments were submitted to double blind histopathological evaluation and the number of megakaryocytes was counted. Besides, animals' serum was measured after 49days of infection, and cytokines (IFN-γ, IL-4, IL-10, IL-12), as well as the Th1/Th2 correlation (IFN-γ/IL-4 and IFN-γ/IL-10), were analyzed. Spleen histological parameters were classified as: healthy appearance (G1); discreet (G3-G7), moderate (G2) and moderate to severe (G8) white pulp hyperplasia; proliferation of megakaryocytes (G2-G8), and intense disruption (G2-G8). All groups, except for G7, showed higher percentages of megakaryocytes per field ranging from 87% to 15%, when compared to healthy animals (G1). Th1 predominance in IFN-γ/IL-4 ratio (comparing to G2) was detected in G4, G5, G6 and G7. Finally, pre/post (BioLi30x) and post-treatment (Antimonium crudum 30x) presented reduction of megakaryocytes/spleen changes due to immunomodulation animal process, controlling the infection process, probably by the Th1 cytokine predominance.

Polymorphisms of blood forms and in vitro metacyclogenesis of Trypanosoma cruzi I, II, and IV.

Trypanosoma cruzi is the etiologic agent of American trypanosomiasis has broad biological and genetic diversity. Remaining to be studied are polymorphisms of the blood forms and metacyclogenesis of different T. cruzi discrete typing units (DTUs). Our goal was to evaluate the relationship between T. cruzi DTUs, the morphology of blood trypomastigotes, and in vitro metacyclogenesis. T. cruzi strains that pertained to DTUs TcI, TcII, and TcIV from different Brazilian states were used. Parameters that were related to the morphology of eight strains were assessed in thin blood smears that were obtained from mice that were inoculated with blood or culture forms, depending on strain. The metacyclogenesis of 12 strains was measured using smears with Liver Infusion Tryptose culture medium and M16 culture medium (which is poor in nutrients and has a low pH) at the exponential phase of growth, both stained with Giemsa. The morphological pattern of TcII strains was consistent with broad forms of the parasite. In TcIV strains, slender forms predominated. The Y strain (TcII) was morphologically more similar to TcIV. Significant differences in polymorphisms were observed between DTUs. Metacyclogenesis parameters, although displaying large standard deviations, differed between the DTUs, with the following descending rank order: TcII > TcI > TcIV. The mean numbers of metacyclic trypomastigotes for TcII were significantly higher than the other DTUs. Although the DTUs presented overlapping characteristics, the general pattern was that different DTUs exhibited significantly different morphologies and metacyclogenesis, suggesting that the genetic diversity of T. cruzi could be related to parameters that are associated with the evolution of infection in mammalian hosts and its ability to disperse in nature.

Histopathological lesions in encephalon and heart of mice infected with Toxoplasma gondii increase after Lycopodium clavatum 200dH treatment.

In many cases, symptoms of toxoplasmosis are mistaken for the ones of other infectious diseases. Clinical signs are rare in immunocompetent people. However, when they arise, in the acute phase of infection, several organs are affected due to the rapid spread of tachyzoites through the bloodstream. In the present study, the reduction of tachyzoites in peripheral blood of mice of G72 (infected 72h after treatment) and G48 (infected 48h after treatment and treated three more times), when compared with IC (infected and non-treated), suggests protective effect exerted by Lycopodium clavatum. If on the one hand L. clavatum brought benefits, reducing parasitemia, on the other hand, the parasitism became exacerbated. Histopathological analysis demonstrated focal, multifocal and diffuse inflammatory infiltrates, ranging from absent, discreet, moderate to intense, in heart and encephalon of mice of NIC (non-infected and non-treated), IC, G48 and G72 groups, respectively. In the perivascular region and meninges, the injuries were enlarged. The presence of tachyzoites was demonstrated through immunohistochemical (IHC) assay in myocardium. Toxoplasma gondii induced increase of collagen fibers in myocardium of mice of G72 and G48 groups, compared with IC (p<0.05) and NIC (p<0.001). The presence of inflammatory infiltrates, as well as the progressive fibrosis, caused myocardial remodeling in animals treated with L. clavatum. Counterstaining with H&E suggests TGF-ß expression by mononuclear cells in the inflammatory infiltrate. Based on our results, we can conclude that the adopted regimen and potency exerted a protective effect, reducing parasitemia. However, it intensified the histopathological lesions in encephalon and heart of mice infected with T. gondii.

Biotherapic 200dH is harmful to acute murine infection with Trypanosoma cruzi

Biotherapics employed to treat mice infected by Trypanosoma cruzi were carried out with encouraging results. The aim of this study was evaluated the effect of biotherapic of Trypanosoma cruzi 200dH, using two different schedules of treatment. Swiss male mice, aged 56 days-old were infected intraperitonealy with 1,400 blood trypomastigotes of Trypanosoma cruzi Y strain and were divided into groups: C.I.- infected animals, E.D. â€" Infected animals treated from the day 1 until the end of the experiment; 200dH S.D. â€" Infected animals treated on the day 1. Parasitological, clinical and immunological parameters were evaluated. The group of animals that received the medicine in a single dose presented higher value to total parasitaemia and lower value of pre patent period compared to control untreated group (p<0.05), as well as the number of amastigotes which was also higher for this group (S.D.) (p<0.05). Clinically, the S.D.group presented more stable temperature (p<0.05) but not presented another clinical difference among treatments. IL-6 and TNF-α presented similar dosage among treated groups as well as IL-4 and IL-10. IL-17A and INF-γ, presented highest values to S.D. group (p<0.05). All animals died until the 20th day of infection. The lack of improvement in clinical and parasitological parameters, the untimely death and the immunological imbalance display the harmful evolution of the experimental infection by T. cruzi using biotherapic 200dH. The results could be useful for homeopathic physicians. In human clinical use, the choice of dynamizations and treatment schedule should consider acute and chronic diseases to achieve the expected results. (AU)

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.

Predominance of Th1 response, increase of megakaryocytes and Kupffer cells are related to survival in Trypanosoma cruzi infected mice treated with Lycopodium clavatum.

We investigated the number of megakaryocytes, Kupffer cells and ratios of Th1/Th2 and Th1/Th17 cytokines in survival of mice infected with Y strain of Trypanosoma cruzi and treated with Lycopodium clavatum. In a blind, randomized and controlled assay, Swiss male mice, 8weeks-old, infected with 1400 trypomastigotes (Y strain) were divided into groups and treated with: GLy - Lycopodium clavatum dynamization13c and GCI - alcohol solution 7° GL (vehicle medicine). The treatment was offered two days before infection and on the 2nd, 4th and 6th days after infection, overnight (1mL/100mL) and ad libitum. Parameters assessed were: survival rate, number of megakaryocytes and Kupffer cells, cytokines dosage (TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17), Th1/Th2 and Th1/Th17 ratios. The increase in megakaryocytes, Kupffer cells, predominance of Th1 response, with increased TNF-α, IL-10, TNF-α/IL-4, TNF-α/IL-17 and decreased IL-6 IL-6/IL-4, are related to increased survival in mice infected with T. cruzi and treated with Lycopodium clavatum 13c. This result demonstrates the possibility of an alternative approach for the treatment of Chagas disease with dynamized drugs.

Trypanosoma cruzi (CEPA CL) promove perda neuronal no plexo mioentérico do cólon de ratos / Trypanosoma cruzi (STRAIN CL) causes neuronal loss in rats´ myo-enteric plexus of the colon

Trypanosoma cruzi, um protozoário parasita, é o agente etiológico da doença de Chagas. A infecção chagásica apresenta duas fases: aguda e crônica. Na fase aguda a mortalidade é mais frequente, sendo que o organismo que consegue sobreviver a ela passa para a fase crônica indeterminada. No trato gastrointestinal o T. gondii pode parasitar o sistema nervoso intramural formando os megaesôfago e o megacólon. Não existem trabalhos que relacionam a perda neuronal do plexo mientérico com a infecção com a cepa CL do T. cruzi. Portanto, o objetivo deste trabalho foi verificar alterações morfoquantitativas no plexo mioentérico no colo distal de ratos infectados com a cepa CL durante a fase aguda e crônica da doença. Foram utilizados 20 ratos divididos em quatro grupos: infectado com a cepa CL durante 7 dias (grupo IA) do T. cruzi e seu controle (grupo CA), ambos sacrificados após sete dias do início do experimento, outro grupo infectado (IC) e sacrificado após trinta dias e seu controle (CC). Foi realizada a estimativa da densidade neuronal e a morfometria das áreas do perfil dos corpos celulares dos neurônios através da técnica de Giemsa. A análise quantitativa demonstrou uma redução significativa no número de neurônios nos grupos infectados IA e IC. Em relação ao perfil neuronal foi observada uma atrofia dos neurônios do grupo IC em relação ao controle. Portanto, a cepa CL do T. cruzi provoca uma denervação do plexo mioentérico do colo sem contudo ocasionar hipertrofia neuronal no tempo experimental de trinta dias.

The parasite protozoa Trypanosoma cruzi is the etiological agent of Chagas´s disease. Infection comprises the acute and chronic type. Mortality in the former is more frequent and the organism that survives passes to the undetermined chronic phase. T. gondii within the gastrointestinal tract parasites the intramural nervous system and causes mega-esophagus and mega-colon. No reports in the literature relate neuronal loss of the myo-enteric plexus with infection by strain CL of T. cruzi. Current analysis verifies morphoquantitative alterations in the myo-enteric plexus in rats´ distal colon infected with strain CL during the acute and chronic phase of the disease. Twenty rats were divided into four groups: infected with strain CL of T. cruzi during 7 days (Group IA) and its control (Group CA), killed after seven days from the start of the experiment; another infected group (IC), killed after 30 days and its control (CC). Neuronal density and morphometry of the areas of cell bodies of neurons were estimated by Giemsa technique. Quantitative analysis showed a significant decrease in the number of neurons in infected groups IA and IC. In the case of the neuronal profile, atrophy of neurons of group IC was reported when compared to control. Strain CL of T. cruzi causes a de-nerving of the myo-enteric plexus of the colon without causing a neuronal hypertrophy during the thirty days of experimental period.

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.