Oxfendazole induces protein catabolism and gluconeogenesis in experimental neurocysticercosis.

Neurocysticercosis (NCC) is an endemic public health disease of the central nervous system highly related to epilepsy and seizures. Taenia crassiceps is an experimental model used for NCC and biochemical studies of the host-parasite relationship. For the past 50 years the NCC therapeutic treatment is performed with albendazole (ABZ) and praziquantel which opens a gap for new therapies due to parasitic resistance and other adverse effects of the drugs. Oxfendazole (OXF) is an albendazole derivative with efficacy against tissue cestodes of veterinary importance. The aim of this study was to determine the metabolic impact of OXF on T. crassiceps cysticerci intracranially inoculated in Balb/C mice. The animals were intracranially inoculated with T. crassiceps cysticerci and 30 days later received single dose oral treatment of OXF, ABZ and NaCl 0.9% (control group). The metabolic impact was quantified through the detection of metabolites from glycolysis, anaerobic fermentation of lactate and propionate, tricarboxylic acid cycle, protein catabolism, fatty acids oxidation. The differences observed in the concentrations of metabolites from the OXF treated group showed that the drug induced gluconeogenesis, increase in protein catabolism, fatty acids oxidation and propionate fermentation in comparison to the ABZ and control treated groups. In conclusion, OXF induced greater metabolic impact in T. crassiceps cysticerci than the standard NCC treatment, ABZ, showing that it may represent an alternative drug for its treatment.

In Vivo Treatment with the Combination of Nitazoxanide and Flubendazole Induces Gluconeogenesis and Protein Catabolism in Taenia crassiceps cysticerci.

PURPOSE: Cysticercosis is the presence of Taenia solium larvae in humans or swines tissues. It is a public health problem related to bad hygienic habits and consumption of infected pork. T. crassiceps is a widely used cysticercosis experimental model. The combination of two effective drugs such as nitazoxanide (NTZ) and flubendazole (FBZ) may potentialize their effect. The aim of this study was to use biochemical analysis to determine the metabolic impact of the combination of NTZ and FBZ on cysticerci inoculated intraperitoneally in mice. METHODS: Balb/c mice intraperitoneally infected with T. crassiceps cysticerci received a single oral dose NTZ/FBZ (50 mg/kg). 24 h after the treatment the cysticerci were removed, frozen and analyzed by high performance liquid chromatography regarding the detection of the following metabolic pathways: glycolysis, gluconeogenesis, homolactic fermentation, tricarboxylic acid cycle, proteins catabolism and fatty acids oxidation. RESULTS: The treatment with the drugs combination induced a statistically significant increase in gluconeogenesis and in protein catabolism when compared to the control groups. CONCLUSION: The drugs combination is potentialized and capable of causing greater metabolic stress than the separate treatment with NTZ or FBZ, showing its potential for an alternative cysticercosis treatment.

Histopathologic evaluation of experimental murine neurocysticercosis after treatment with albendazole/nitazoxanide combination.

Neurocysticercosis (NCC) is the most common helminthic brain infection related to epilepsy. Only albendazole (ABZ) and praziquantel are used in its treatment. The development of new therapeutics has been encouraged. Taenia crassiceps cysticerci intracranial infection is the experimental model used in NCC studies. This study evaluated the histopathology of the brains of BALB/c mice experimentally infected with T. crassiceps cysticerci after the treatment with the ABZ/nitazoxanide (NTZ) combination. Thirty days after the inoculation the mice received an oral single dose of the ABZ/NTZ combination (40 mg kg-1 each). The control groups were treated with: NaCl 0.9%; ABZ or NTZ. The histopathologic evaluation of the brains was performed 24 h after treatment. The ABZ treatment induced discrete mononuclear inflammatory infiltration, meningitis, gliosis, hyperaemia and hippocampus compression; moderate ependimitis and oedema. The NTZ treatment induced accentuated inflammatory infiltration, foamy macrophages, ependimitis, choroiditis, gliosis and hyperaemia and moderate oedema. The ABZ/NTZ combination treatment induced a significant decrease in the polymorphonuclear inflammatory infiltration, ependimitis, choroiditis, gliosis, hyperaemia and ventriculomegaly in comparison with the other groups. The cysticerci showed destruction of the tegument not observed in other groups. The ABZ/NTZ combination is efficient as the parasite showed signs of destruction and lower damage to the host's tissue.

Metabolic effects of anthelminthic drugs in the larval stage of the cestode Taenia crassiceps, cysticercosis experimental model - A review.

Taenia crassiceps is an experimental model used for cysticercosis studies and has suffered metabolic analyzes regarding the effect of anthelminthic drugs. The metabolic analyses are useful tools to determine the drugs mode of action and the parasite`s survival mechanisms. The energetic pathways are good candidates for this kind of approach as they are essential for the parasite`s survival and adaptation to the environment. In this review we discuss the anthelminthic drugs mode of action and its metabolic impact on Taenia crassiceps cysticerci.

Partial inhibition of the tricarboxylic acid cycle in Taenia crassiceps cysticerci after the in vitro exposure to a benzimidazole derivative (RCB15).

The benzimidazole derivative, 6-chloro-5-(2,3-dichlorophenoxy)-2-(trifluoromethyl)-1H-benzimidazole (RCB15), has a similar mode of action and efficacy as albendazole, a commonly used anthelminthic drugs. The aim of this study was to evaluate its influence on the tricarboxylic acid cycle in Taenia crassiceps cysticerci. The parasites were cultured in supplemented RPMI medium containing albendazole sulfoxide (ABZSO) or RCB15, for 24 h. Then, frozen in liquid nitrogen for organic metabolites extraction. Samples were analyzed by high performance liquid chromatography and organic acids of the tricarboxylic acid cycle were detected. It was possible to observe changes in the concentrations of all acids involved in this metabolic pathway, with the exception of α-ketoglutarate, which was not detected in the control group neither in most of the treated groups. It indicates that the parasite presented a partial inhibition of the tricarboxylic acid cycle. The significant increase in the concentration of citrate, oxaloacetate and succinate in the RCB15 treated groups may indicate an activation of the fumarate reductase pathway, leading to metabolic distress. Therefore RCB15 may be considered an alternative for the treatment of tissue parasitic diseases, since it induced changes in the main metabolic pathway of the parasite.

In vitro nitazoxanide exposure affects energetic metabolism of Taenia crassiceps.

Nitazoxanide (NTZ) is a broad-spectrum drug used in intestinal infections, but still poorly explored in the treatment of parasitic tissular infections. This study aimed to evaluate the in vitro responses of the energetic metabolism of T. crassiceps cysticerci induced by NTZ. The organic acids of the tricarboxylic acid cycle, products derived from fatty acids oxidation and protein catabolism were analyzed. These acids were quantified after 24 h of in vitro exposure to different NTZ concentrations. A positive control group was performed with albendazole sulfoxide (ABZSO). The significant alterations in citrate, fumarate and malate concentrations showed the NTZ influence in the tricarboxylic acid (TCA) cycle. The non-detection of acetate confirmed that the main mode of action of NTZ is effective against T. crassiceps cysticerci. The statistical differences in fumarate, urea and beta-hydroxybutyrate concentrations showed the NTZ effect on protein catabolism and fatty acid oxidation. Therefore, the main energetic pathways such as the TCA cycle, protein catabolism and fatty acids oxidation were altered after in vitro NTZ exposure. In conclusion, NTZ induced a significant metabolic stress in the parasite indicating that it may be used as an alternative therapeutic choice for cysticercosis treatment. The use of metabolic approaches to establish comparisons between anti parasitic drugs mode of actions is proposed.

Photobiomodulation associated to cellular therapy improve wound healing of experimental full thickness burn wounds in rats.

Adipose derived stromal vascular fraction (SVF) is a method of cell therapy potentially applicable for treatment of full thickness burns. Here we investigated if the association of photobiomodulation (PBM) with SVF therapy could improve wound healing in experimentally induced full thickness burn wounds in rats compared to the topical agent 2% silver sulfadiazine in a dose-dependent manner. Sixty-six male Wistar rats were divided in 4 groups containing 5 animals each which received the following treatments: 2% sulfadiazine (SD), SVF, SVF plus PBM at 30 mW (SVFL30), and SVF plus PBM at 100 mW (SVFL100). Two donor animals were used for each experimental series with 3, 7 and 30 days. Digital photography, microscopic analysis with Hematoxilin and Eosin (H&E), quantification of collagen type I by picrosirius red staining analysis and wound contraction evaluation were performed in order to quantify the results. At day 3 SVF alone or combined with PBM promoted increased early inflammatory response compared to SD. At day 7 SVFL30 and SVFL100 enhanced inflammatory cells infiltration, angiogenesis and fibroblast content compared to SVF and SD groups. At day 30 collagen concentration and wound contraction were higher in SVFL30 when compared to the other groups. In conclusion PBM promotes a synergistic outcome with SVF therapy with a dose dependent effect potentializing wound healing of experimental full thickness burns in rats through amplification of early inflammatory response, enhanced angiogenesis, fibroblast content, accentuated wound contraction and collagen concentration.

Oral nitazoxanide treatment of experimental neurocysticercosis induces gluconeogenesis in Taenia crassiceps cysticerci.

Neurocysticercosis is the most frequent helminthiasis of the central nervous system and is caused by the presence of Taenia solium cysticerci. Nitazoxanide (NTZ) is an antifolate containing the pyrrolopyrimidine-based nucleus that exerts its antiprotozoal activity due to interference with the pyruvate:ferredoxin oxidoreductase (PFOR) enzyme which is essential to anaerobic energy metabolism. The aim of this work was to determine the effect of NTZ on the energetic metabolism of Taenia crassiceps cysticerci intracranially inoculated BALB /c mice. The infected animals were treated with a single oral dose of NTZ 30 days after the inoculation. Analysis of the organic acids was performed through high performance liquid chromatography. Glucose was detected only in the treated groups, alongside with a significant decrease in lactate, pyruvate and oxaloacetate concentrations which indicate an increase in gluconeogenesis. The non-detection of alpha-ketoglutarate indicated the use of the fumarate reductase pathway in all groups. It was possible to confirm the drugs mode of action due to the non-detection of acetate in the treated groups. There was an increase in the fatty acids oxidation. Therefore it was possible to observe that NTZ induces gluconeogenesis as well as the increase of alternative energetic pathways such as fatty acids oxidation in T. crassiceps cysticerci.

Taenia crassiceps antigens induce a Th2 immune response and attenuate injuries experimentally induced by neurotoxoplasmosis in BALB/c mice.

Toxoplasma gondii is a pathogenic agent responsible for causing both systemic and local disease which elicits a typically pro-inflammatory, Th1 immune response. Taenia crassiceps antigen induces a Th2 immune response that immunomodulates Th1 based infections. Therefore the aim of this study was to evaluate whether T. crassiceps cysticerci antigens are able to modulate the inflammatory response triggered in experimental neurotoxoplasmosis (NT). BALB/c mice were inoculated with T. gondii cysts and/or cysticerci antigens and euthanized at 60 and 90days after inoculation (DAI). The histopathology of the brains and cytokines produced by spleen cells culture were performed. The animals from the NT group, 90DAI (NT90), presented greater intensity of lesions such as vasculitis, meningitis and microgliosis and cytokines from Th1 profile characterized by high levels of IFN-gamma. While in the T. crassiceps antigens group, 60DAI, there were more discrete lesions and high levels of IL-4, a Th2 cytokine. In the NT co-inoculated with cysticerci antigens group the parenchyma lesions were more discrete with lower levels of IFN-gamma and higher levels of IL-4 when compared to NT90. Therefore the inoculation of T. crassiceps antigens attenuated the brain lesions caused by T. gondii inducing a Th2 immune response.

Nitazoxanide induces in vitro metabolic acidosis in Taenia crassiceps cysticerci.

Nitazoxanide (NTZ) is a broad-spectrum anti-parasitic drug used against a wide variety of protozoans and helminthes. Albendazole, its active metabolite albendazole sulfoxide (ABZSO), is one of the drugs of choice to treat both intestinal and tissue helminth and protozoan infections. However little is known regarding their impact on the metabolism of parasites. The aim of this study was to compare the in vitro effect of NTZ and ABZSO in the glycolysis of Taenia crassiceps cysticerci. The cysticerci were treated with 1.2; 0.6; 0.3 or 0.15 µg/mL of NTZ or ABZSO. Chromatographic and spectrophotometric analyses were performed in the culture medium and in the cysticerci extract. Regarding the glucose concentrations was possible to observe two responses: impair of the uptake and gluconeogenesis. The pyruvate concentrations were increased in the ABZSO treated group. Lactate concentrations were increased in the culture medium of NTZ treated groups. Therefore it was possible to infer that the metabolic acidosis was greater in the group treated with NTZ than in the ABZSO treated group indicating that this is one of the modes of action used by this drug to induce the parasite death.

IFN-gamma role in granuloma formation in experimental subcutaneous cysticercosis.

Cysticercosis is an infection caused by the metacestode larval stage of Taenia parasites in tissues and elicits a host-parasite reaction in which the immune response may be decisive in the disease development. The aim of this study was to evaluate the role of IFNγ (IFN-gamma) in the experimental model of subcutaneous infection with Taenia crassiceps (T. crassiceps) cysticerci using IFNγ knockout mice. Male C57BL/6 and C57BL/6 KO IFNγ mice 8-12 weeks of age were inoculated with T. crassiceps cysticerci into the subcutaneous tissue of the dorsum. At 7 and 30 (acute phase), 60 and 90 (chronic phase) days post infection, animals from each group had their blood and the subcutaneous tissues collected for serologic and pathological studies. IFNγ and IL-4 were dosed and the histopathological analysis was performed. In the presence of IFNγ there was the establishment of a mixed Th1/Th2 systemic immune profile. This profile also locally induced the granuloma formation which was constituted by cells that played important roles in the parasitary destruction and that were likely associated to the Th1 axis of mixed immune response. On the other hand, the absence of IFNγ appears to favor the parasitary growth which may be related to the development of a systemic Th2 immune response. This profile influenced the granuloma formation with immunoregulatory properties and appears to be important in the collagen synthesis.

Activity of low level laser therapy on burning wounds in diabetic rats / Atividade terapêutica do laser de baixa intensidade em queimaduras em ratos diabéticos / Actividad terapéutica de láser de baja intensidad en quemaduras de ratones diabéticos

This study evaluated the effects of low level laser (LLL) therapy in the healing of third degree burning wounds in diabetic and non-diabetic Wistar rats. Methods: The diabetes was experimentally induced with streptozotocine 14 days before the burning injury induction. The rats suffered the induction of third-degree burning injury and were divided into four groups as follows: control group; non-diabetic treated group; diabetic group; diabetic treated group. All animals received occlusive bandages during the experimental days. The treated animals received the following treatment in alternate days: diode GaAlAs laser (650 nm/ 12 mW), fluency of 3 J/cm2 until the 7th experimental day followed by 6 J/cm2 from the 7th day until the euthanasia day. The burning wounds were morphometrically, macroscopically and microscopically evaluated at 3, 7, 14, 21 and 30 days after the induction. Results: The wound contraction was significantly higher in all experimental days in treated groups when compared to the diabetic and non-diabetic control groups. Microscopically, there was a significant increase in angiogenesis and in fibrogenesis during the proliferative stage in the treated groups. Conclusion: Therefore, we conclude that LLL therapy favored the tissue healing process with 3 J/cm2 dosage for the inflammatory stage and with 6 J/cm2 dosage for the proliferative and remodeling ones, accelerating the burning wound contraction and improving the healing process...

Este estudo avaliou os efeitos da terapia a laser de baixa intensidade (LBI) na cicatrização de feridas por queimadura de terceiro grau em ratos Wistar diabéticos e não diabéticos. Métodos: A diabete foi induzida experimentalmente com estreptozotocina 14 dias antes da indução da lesão por queimadura. Os ratos sofreram a indução da lesão por queimadura de terceiro grau e foram divididos em quatro grupos: grupo controle; grupo tratado não diabético; grupo diabético; grupo tratado diabético. Todos os animais receberam curativos oclusivos durante os dias experimentais. Os animais tratados receberam o seguinte tratamento em dias alternados: laser diodos GaAIAs (650 nm/12 mW), fluência de 3 J/cm2 até o sétimo dia experimental, seguido por 6 J/cm2 a partir do sétimo dia até ao dia eutanásia. As feridas por queimaduras foram avaliadas morfometricamente, macro e microscopicamente em 3, 7, 14, 21 e 30 dias após a indução. Resultados: A contração da ferida foi significativamente maior em todos os dias experimentais nos grupos tratados quando comparados com os grupos controle diabéticos e não diabéticos. Microscopicamente, houve aumento significativo na angiogênese e na fibrogênese durante a fase proliferativa nos grupos tratados. Conclusões: Concluímos que a terapia LBI favoreceu o processo de cicatrização do tecido com dosagem de 3 J/cm2 na fase inflamatória e com dosagem de 6 J/cm2 nas fases proliferativa e de remodelação, acelerando a contração da ferida por queimadura e melhorando o processo de cicatrização. DESCRITORES: Queimaduras. Diabetes Mellitus. Terapia com Luz de Baixa Intensidade. Patologia...

Este estudio evaluó los efectos de la terapia láser de baja intensidad (LBI) en la cicatrización de heridas por quemadura de 3er. grado en ratones Wistar diabéticos y no diabéticos. Métodos: La diabetes fue inducida experimentalmente con Estreptozotocina 14 días antes de la inducción de la lesión por quemadura. Después de la inducción de la lesión por quemadura de 3er. grado, los ratones fueron divididos en cuatro grupos: control, tratado no-diabético, diabético y tratado-diabético. Todos los animales recibieron curativos oclusivos durante los días de los experimentos. Los animales tratados recibieron el siguiente tratamiento en días alternados: láser diodos GaAIAs (650 nm/12 mW), flujo de 3 J/cm2 hasta el séptimo dia del experimento, seguido por 6 J/cm2 a partir del séptimo día hasta el día de la eutanasia. Las heridas por quemadura fueron evaluadas morfométricamente, macro y microscópicamente en 3, 7, 14, 21 y 30 días después de la inducción. Resultados: La contracción de la herida fue significativamente mayor en todos os días de experimento en los grupos tratados cuando comparados con los grupos control diabéticos y no-diabéticos. Microscopicamente, hubo aumento significativo en la angiogénesis y en la fibrogénesis durante la fase proliferativa en los grupos tratados. Conclusiones: Concluimos que la terapia LBI favoreció el proceso de cicatrización del tejido con dosis de 3 J/cm2 en la fase inflamatoria y con dosis de 6 J/cm2 en las fases proliferativa y de remodelación, acelerando la contracción de la herida por quemadura y mejorando el proceso de cicatrización...

Cellular immune response in intraventricular experimental neurocysticercosis.

Neurocysticercosis (NCC) is considered a neglected parasitic infection of the human central nervous system. Its pathogenesis is due to the host immune response, stage of evolution and location of the parasite. The aim of this study was to evaluate the in situ and systemic immune response through cytokines dosage (IL-4, IL-10, IL-17 and IFN-γ) as well as the local inflammatory response of the experimental NCC with Taenia crassiceps. The in situ and systemic cellular and inflammatory immune response were evaluated through the cytokines quantification at 7, 30, 60 and 90 days after inoculation and histopathological analysis. All cysticerci were found within the cerebral ventricles. There was a discrete intensity of inflammatory cells of mixed immune profile, polymorphonuclear and mononuclear cells, at the beginning of the infection and predominance of mononuclear cells at the end. The systemic immune response showed a significant increase in all the analysed cytokines and predominance of the Th2 immune profile cytokines at the end of the infection. These results indicate that the location of the cysticerci may lead to ventriculomegaly. The acute phase of the infection showed a mixed Th1/Th17 profile accompanied by high levels of IL-10 while the late phase showed a Th2 immune profile.

In silico search of energy metabolism inhibitors for alternative leishmaniasis treatments.

Leishmaniasis is a complex disease that affects mammals and is caused by approximately 20 distinct protozoa from the genus Leishmania. Leishmaniasis is an endemic disease that exerts a large socioeconomic impact on poor and developing countries. The current treatment for leishmaniasis is complex, expensive, and poorly efficacious. Thus, there is an urgent need to develop more selective, less expensive new drugs. The energy metabolism pathways of Leishmania include several interesting targets for specific inhibitors. In the present study, we sought to establish which energy metabolism enzymes in Leishmania could be targets for inhibitors that have already been approved for the treatment of other diseases. We were able to identify 94 genes and 93 Leishmania energy metabolism targets. Using each gene's designation as a search criterion in the TriTrypDB database, we located the predicted peptide sequences, which in turn were used to interrogate the DrugBank, Therapeutic Target Database (TTD), and PubChem databases. We identified 44 putative targets of which 11 are predicted to be amenable to inhibition by drugs which have already been approved for use in humans for 11 of these targets. We propose that these drugs should be experimentally tested and potentially used in the treatment of leishmaniasis.

Energetic metabolism of axenic promastigotes of Leishmania (Viannia) braziliensis.

Leishmania spp are protozoans capable of carbohydrates degradation and as energy source they can use glucose, aminoacids or lipids from the environment. The products of the metabolic pathways such as organic acids may be used as an index of their energetic metabolic profile. Therefore, in this study a metabolic profile comparison was made between promastigotes from one reference strain (MHOM/BR/1975/M2903) and two different isolates of Leishmania (Viannia) braziliensis (MHOM/BR/2003/IMG3 and MHOM/BR/2005/RPL5). The parasites culture was performed in complete Grace's culture media seeded in 24-well plates at 26°C. During the growth curve performance samples were collected from the logarithmic and stationary phases of culture and therefore analyzed by high performance liquid chromatography (HPLC) and spectrophotometry assays to determine the concentrations of glucose, lactate, citrate, α-ketoglutarate, succinate, fumarate, malate, oxaloacetate and ß-hydroxybutirate which are indicative of the energetic pathways. It was possible to detect an increase in the glucose from the stationary phase from the M2903 strain when compared to the logarithmic phase while in the IMG3 and RPL5 isolates there was a decrease (p<0.05). The spectrophotometric and chromatographic results indicated that the logarithmic phase which presents higher energy consumption due to the intense replication rate have the energetic pathways intensified. It was also possible to note some metabolic differences between the analyzed parasites which may indicate possible adaptations of the parasite when facing different environmental and physiological conditions during its life cycle and that these differences may help in the understanding of the diversity of the host-parasite relationship from Leishmania parasites.

Morphological alterations and growth inhibition of Leishmania (L.)amazonensis promastigotes exposed to zidovudine (AZT).

Leishmania parasites cause a worldwide public health disease and its treatment is still based on pentavalent antimonials which present financial and toxicologic limitations. Some nucleosidic derivatives have demonstrated anti-leishmanial properties and this study aims to evaluate the in vitro morphologic alterations and growth inhibition of Leishmania (L.) amazonensis promastigotes exposed to zidovudine at several concentrations. The citotoxicity of zidovudine (AZT) to macrophages was determined by an MTT assay. After which the promastigotes were exposed to concentrations of AZT, ranging from 1 to 50 µM. The evaluation of survival and morphometry alterations were performed in two distinct phases of in vitro growth, on the third and sixth days, representing the logarithmic and stationary phases, respectively. Slides with the promastigotes were photographed and analyzed using Image J. A significant reduction of parasite number in the logarithmic phase of in vitro growth was observed when the parasites were submitted to 20, 30, 40, and 50 µM of AZT. Morphometric alterations were observed such as an increase in width of the body, cytoplasmic granulations and vacuolizations. These data indicate the toxicity of AZT which prevents the parasite's multiplication, indicating a promising use of AZT as an anti-leishmania drug.

Taenia crassiceps organic acids detected in cysticerci.

Taenia crassiceps cysticerci is used as an experimental model to cysticercosis studies; however there are subcutaneous cases of cysticercosis caused by these cysticerci. It remains unclear in the literature the energetic and fatty acid metabolism in cestodes. Its metabolic study may provide knowledge of pathways that may serve as potential anti-helminthic drugs sites of action. In this work we studied the citric acid cycle organic acids and the fatty acid oxidation in cysticerci removed from mice with 21 and 42 days of infection in two different evolutive stages: growing and final. The organic acids were extracted using perchloric acid and analyzed by HPLC methodology. We found significant statistically differences in oxalate, malate, lactate, and beta-hydroxybutirate concentrations between cysticerci. These results indicate the aerobic metabolism in vivo in spite of the low oxygen concentration of its habitat, and also indicate the presence of fatty acid oxidation as an alternative energetic source.