Trypanosoma cruzi: Does the intake of nanoencapsulated benznidazole control acute infections?

Chagas Disease (CD) affects around eight million people worldwide. It is considered a neglected disease that presents few treatment options with efficacy only in the acute phase. Nanoparticles have many positive qualities for treating parasite infections and may be effectively and widely employed in clinical medicine. This research aimed to evaluate the nanoencapsulated benznidazole treatment in animals experimentally infected with Trypanosoma cruzi. To analyze the treatment efficacy, we evaluated survival during thirty days, parasitemia, genotoxicity, and heart and liver histopathology. Thirty-five female Swiss mice were organized into seven groups characterizing a dose curve: A - Negative control (uninfected animals), B - Positive control (infected animals), C - Benznidazole (BNZ) 100 mg/kg (infected animals), D - 5 mg/kg Benznidazole nanocapsules (NBNZ) (infected animals), E - 10 mg/kg Benznidazole nanocapsules (infected animals), F - 15 mg/kg Benznidazole nanocapsules (infected animals), G - 20 mg/kg Benznidazole nanocapsules (infected animals). The animals were infected with the Y strain of T. cruzi intraperitoneally. The treatment was administered for eight days by oral gavage. It was possible to observe that the treatment with the highest NBNZ dose presented efficacy similar to the standard benznidazole drug. The 20 mg/kg NBNZ dose was able to reduce parasitemia, increase survival, and drastically reduce heart and liver tissue damage compared to the 100 mg/kg BNZ dose. Moreover, it showed a lower DNA damage index than the BNZ treatment. In conclusion, the nanoencapsulation of BNZ promotes an improvement in parasite proliferation control with a five times smaller dose relative to the standard dose of free BNZ, thus demonstrating to be a potential innovative therapy for CD.

Branchial bioenergetics dysfunction as a relevant pathophysiological mechanism in freshwater silver catfish (Rhamdia quelen) experimentally infected with Flavobacterium columnare.

Flavobacterium columnare, the causative agent of columnaris disease, is a serious bacterial disease responsible for causing devastating mortality rates in several species of freshwater fish, leading to severe economic losses in the aquaculture industry. Notwithstanding the enormous impacts this disease can have, very little is known regarding the interaction between the host and bacterium in terms of the mortality rate of silver catfish (Rhamdia quelen), as well its linkage to gill energetic homeostasis. Therefore, we conducted independent experiments to evaluate the mortality rates caused by F. columnare in silver catfish, as well as whether columnaris disease impairs the enzymes of the phosphoryl transfer network in gills of silver catfish and the pathways involved in this inhibition. Experiment I revealed that clinical signs started to appear 72 h post-infection (hpi), manifesting as lethargy, skin necrosis, fin erosion and gill discoloration. Silver catfish began to die at 96 hpi, and 100% mortality was observed at 120 hpi. Experiment II revealed that creatine kinase (CK, cytosolic and mitochondrial) and pyruvate kinase (PK) activities were inhibited in silver catfish experimentally infected with F. columnare, while no significant difference was observed between experimental and control groups with respect to adenylate kinase activity. Activity of the branchial sodium-potassium pump (Na+, K+-ATPase) was inhibited while reactive oxygen species (ROS) and lipid peroxidation levels were higher in silver catfish experimentally infected with F. columnare than in the control group at 72 hpi. Based on these data, the impairment of CK activity elicited by F. columnare caused a disruption in branchial energetic balance, possibly reducing ATP availability in the gills and provoking impairment of Na+, K +ATPase activity. The inhibition of CK and PK activities appears to be mediated by ROS overproduction and lipid peroxidation, both of which contribute to disease pathogenesis associated with branchial tissue.

Purinergic signaling creates an anti-inflammatory profile in spleens of grass carp Ctenopharyngodon idella naturally infected by Saprolegnia parasitica: An attempt to prevent ATP pro-inflammatory effects.

Extracellular adenosine triphosphate (ATP) is as key mediator of immune and inflammatory responses. ATP is normally sequestered in the intracellular milieu and released by apoptotic and necrotic cells, where it acts as a pro-inflammatory mediator in the extracellular milieu. A limited number of studies have explored the involvement of purinergic signaling in oomycete infections, including Saprolegnia parasitica; this is a most destructive oomycete pathogen, associated with high mortality and severe economic losses for fish producers. The aim of this study was to determine whether purinergic signaling exerts anti- or pro-inflammatory effects in spleens of grass carp (Ctenopharyngodon idella) naturally infected by S. parasitica. Animals naturally infected with S. parasitica showed typical gross lesions characterized by cotton-wool tufts on the tail and fins, as well as severe histopathological lesions such as necrosis. Spleen ATP and metabolites of nitric oxide (NOx) levels were higher in fish naturally infected by S. parasitica compared to control on day 7 post-infection (PI). Spleen nucleoside triphosphate diphosphohydrolase (NTPDase) activity (ATP as substrate) was greater in fish naturally infected by S. parasitica than in uninfected on day 7 PI, while no significant differences were observed between groups with respect to NTPDase (adenosine diphosphate as substrate) and 5'-nucleotidase activities. Finally, adenosine deaminase (ADA) activity was lower in fish naturally infected by S. parasitica than in uninfected fish on day 7 PI. In summary, spleen tissue necrosis in the context of saprolegniosis provokes an intense release of ATP into the extracellular milieu, where it interacts with the P2X7 purine receptor and leads to a self-sustained pro-inflammatory deleterious cycle, contributing to an intense inflammatory process. In response to excessive ATP levels in the extracellular milieu, ATP and adenosine hydrolysis were modulated in an attempt to restrict the inflammatory process via upregulation of NTPDase and downregulation of ADA activities. We conclude that the purinergic signaling pathway modulates immune and inflammatory responses during natural infection with S. parasitica.

Oxidative stress and antioxidant responses in Nile tilapia Oreochromis niloticus experimentally infected by Providencia rettgeri.

Bacterial diseases are one of the major problems in freshwater fish culture and have been linked to significant losses and high mortality rate. In this study, Nile tilapia Oreochromis niloticus was infected by Providencia rettgeri to evaluate the oxidative stress and antioxidant responses in the fish tissues. Juvenile Nile tilapia was divided into two groups, as follow: control (uninfected) and experimentally infected with 100 µL of P. rettgeri suspension containing 2.4 × 107 viable cells/fish, and the liver and kidney tissues were collected on days 7 and 14 post-infection (PI). Liver and kidney ROS and lipid peroxidation levels were high in infected fish on day 14 PI compared to control group, while superoxide dismutase activity was lower in liver (days 7 and 14 PI) and kidney (day 14 PI) compared to their respective control groups. Liver and kidney antioxidant capacity against peroxyl radicals, non-proteic, and proteic thiols levels was lower in infected tilapia on day 14 PI compared to control group. Based on these results, P. rettgeri infection may elicit oxidative damage via increased ROS production, decreased ROS elimination and inhibits enzymatic and non-enzymatic antioxidant defense systems; which may contribute directly to disease pathophysiology of infected animals.

Aflatoxin B1-contaminated diet disrupts the blood-brain barrier and affects fish behavior: Involvement of neurotransmitters in brain synaptosomes.

It is known that the cytotoxic effects of aflatoxin B1 (AFB1) in endothelial cells of the blood-brain barrier (BBB) are associated with behavioral dysfunction. However, the effects of a diet contaminated with AFB1 on the behavior of silver catfish remain unknown. Thus, the aim of this study was to evaluate whether an AFB1-contaminated diet (1177 ppb kg feed-1) impaired silver catfish behavior, as well as whether disruption of the BBB and alteration of neurotransmitters in brain synaptosomes are involved. Fish fed a diet contaminated with AFB1 presented a behavioral impairment linked with hyperlocomotion on days 14 and 21 compared with the control group (basal diet). Neurotransmitter levels were also affected on days 14 and 21. The permeability of the BBB to Evans blue dye increased in the intoxicated animals compared with the control group, which suggests that the BBB was disrupted. Moreover, acetylcholinesterase (AChE) activity in brain synaptosomes was increased in fish fed a diet contaminated with AFB1, while activity of the sodium-potassium pump (Na+, K+-ATPase) was decreased. Based on this evidence, the present study shows that silver catfish fed a diet containing AFB1 exhibit behavioral impairments related to hyperlocomotion. This diet caused a disruption of the BBB and brain lesions, which may contribute to the behavioral changes. Also, the alterations in the activities of AChE and Na+, K+-ATPase in brain synaptosomes may directly contribute to this behavior, since they may promote synapse dysfunction. In addition, the hyperlocomotion may be considered an important macroscopic marker indicating possible AFB1 intoxication.

Citrobacter freundii impairs the phosphoryl transfer network in the gills of Rhamdia quelen: Impairment of bioenergetics homeostasis.

The precise coupling of spatially separated intracellular adenosine triphosphate (ATP)-producing and ATP-consuming, catalyzed by creatine kinase (CK), adenylate kinase (AK), and pyruvate kinase (PK), is a critical process in the bioenergetics of tissues with high energy demand, such as the branchial tissue. The effects of Citrobacter freundii infection on gills remain poorly understood, limited only to histopathological studies. Thus, the aim of this study was to evaluate whether experimental infection by C. freundii impairs the enzymes of the phosphoryl transfer network in gills of silver catfish (Rhamdia quelen). The CK (cytosolic and mitochondrial) and AK activities decreased in infected compared to uninfected animals, while the PK activity did not differ between groups. The gill histopathology of infected animals revealed extensive degeneration with fusion and necrosis of secondary lamellae, detachment of superficial epithelium, aneurysm, vessel congestion and inflammatory process. Based on these evidences, the inhibition and absence of an efficient communication between CK compartments caused the impairment of the branchial bioenergetics homeostasis, which was not compensated by the augmentation on branchial AK activity in an attempt to restore energy homeostasis. In summary, these alterations contribute to disease pathogenesis linked to branchial tissue in animals infected with C. freundii.

Purinergic signaling modulates the cerebral inflammatory response in experimentally infected fish with Streptococcus agalactiae: an attempt to improve the immune response.

Appropriate control of the immune response is a critical determinant of fish health, and the purinergic cascade has an important role in the immune and inflammatory responses. This cascade regulates the levels of adenosine triphosphate (ATP), adenosine diphosphate, adenosine monophosphate and adenosine (Ado), molecules involved in physiological or pathological events as inflammatory and anti-inflammatory mediators. Thus, the aim of this study was to evaluate whether purinergic signaling, through the activities of nucleoside triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase, and adenosine deaminase (ADA), is capable of modulating the cerebral immune and inflammatory responses in silver catfish that is experimentally infected with Streptococcus agalactiae. Cerebral NTPDase (with ATP as substrate) and 5'-nucleotidase activities increased, while ADA activity decreased in silver catfish that is experimentally infected with S. agalactiae, compared to the control group. Moreover, the cerebral levels of ATP and Ado increased in infected animals compared to the uninfected control group. Brain histopathology in infected animals revealed inflammatory demyelination (the presence of occasional bubbly collections), increased cellular density in the area near to pia-mater and intercellular edema. Based on this evidence, the modulation of the purinergic cascade by the enzymes NTPDase, 5'-nucleotidase, and ADA exerts an anti-inflammatory profile due to the regulation of ATP and Ado levels. This suggests involvement of purinergic enzymes on streptococcosis pathogenesis, through regulating cerebral ATP and Ado levels, molecules known to participate in physiological or pathological events as inflammatory and anti-inflammatory mediators, respectively. In summary, the modulation of the cerebral purinergic cascade exerts an anti-inflammatory profile in an attempt to reduce inflammatory damage.

Involvement of xanthine oxidase activity with oxidative and inflammatory renal damage in silver catfish experimentally infected with Streptococcus agalactiae: Interplay with reactive oxygen species and nitric oxide.

Xanthine oxidase (XO) is a final enzyme of purine metabolism linked with initiation and progression of infectious diseases, since is considered an important source of reactive oxygen species (ROS) and nitric oxide (NO), developing a pro-oxidant and pro-inflammatory profile in some infectious diseases. Thus, the aim of this study was to evaluate the involvement of XO activity in the renal oxidative and inflammatory damage, as well as the interplay with ROS and metabolites of nitric oxide (NOx) levels in silver catfish experimentally infected with Streptococcus agalactiae. Xanthine oxidase activity, and uric acid, ROS and NOx levels increased in renal tissue of infected animals compared to uninfected animals. Moreover, the histopathological analyses revealed the presence of necrosis, generalized edema and nuclear degeneration of renal tubules. Based on these evidences, the upregulation on renal XO activity exerts a pro-oxidant and pro-inflammatory profile in kidney of fish infected with S. agalactiae. The excessive uric acid levels induced the release of oxidative and inflammatory mediators, such as ROS and NOx, that directly contribute to renal oxidative and inflammatory damage. In summary, the upregulation on XO activity may be considered a pathway involved in the renal injury during S. agalactiae infection.

Streptococcus agalactiae impairs cerebral bioenergetics in experimentally infected silver catfish.

It is becoming evident that bacterial infectious diseases affect brain energy metabolism, where alterations of enzymatic complexes of the mitochondrial respiratory chain and creatine kinase (CK) lead to an impairment of cerebral bioenergetics which contribute to disease pathogenesis in the central nervous system (CNS). Based on this evidence, the aim of this study was to evaluate whether alterations in the activity of complex IV of the respiratory chain and CK contribute to impairment of cerebral bioenergetics during Streptococcus agalactiae infection in silver catfish (Rhamdia quelen). The activity of complex IV of the respiratory chain in brain increased, while the CK activity decreased in infected animals compared to uninfected animals. Brain histopathology revealed inflammatory demyelination, gliosis of the brain and intercellular edema in infected animals. Based on this evidence, S. agalactiae infection causes an impairment in cerebral bioenergetics through the augmentation of complex IV activity, which may be considered an adaptive response to maintain proper functioning of the electron respiratory chain, as well as to ensure ongoing electron flow through the electron transport chain. Moreover, inhibition of cerebral CK activity contributes to lower availability of ATP, contributing to impairment of cerebral energy homeostasis. In summary, these alterations contribute to disease pathogenesis linked to the CNS.

Aeromonas caviae alters the cytosolic and mitochondrial creatine kinase activities in experimentally infected silver catfish: Impairment on renal bioenergetics.

Cytosolic and mitochondrial creatine kinases (CK), through the creatine kinase-phosphocreatine (CK/PCr) system, provide a temporal and spatial energy buffer to maintain cellular energy homeostasis. However, the effects of bacterial infections on the kidney remain poorly understood and are limited only to histopathological analyses. Thus, the aim of this study was to investigate the involvement of cytosolic and mitochondrial CK activities in renal energetic homeostasis in silver catfish experimentally infected with Aeromonas caviae. Cytosolic CK activity decreased in infected animals, while mitochondrial CK activity increased compared to uninfected animals. Moreover, the activity of the sodium-potassium pump (Na+, K+-ATPase) decreased in infected animals compared to uninfected animals. Based on this evidence, it can be concluded that the inhibition of cytosolic CK activity by A. caviae causes an impairment on renal energy homeostasis through the depletion of adenosine triphosphate (ATP) levels. This contributes to the inhibition of Na+, K+-ATPase activity, although the mitochondrial CK activity acted in an attempt to restore the cytosolic ATP levels through a feedback mechanism. In summary, A. caviae infection causes a severe energetic imbalance in infected silver catfish, which may contribute to disease pathogenesis.

Inhibition of the mitochondrial respiratory chain in gills of Rhamdia quelen experimentally infected by Pseudomonas aeruginosa: Interplay with reactive oxygen species.

It has long been recognized that there are several infectious diseases linked to the impairment of enzymatic complexes of the mitochondrial respiratory chain, with consequent production of reactive oxygen species (ROS), that contribute to disease pathogenesis. In this study, we determined whether the inhibition on mitochondrial respiratory chain might be considered a pathway involved in the production of ROS in gills of Rhamdia quelen experimentally infected by P. aeruginosa. The animals were divided into two groups with six fish each: uninfected (the negative control group) and infected (the positive control group). On day 7 post-infection (PI), animals were euthanized and the gills were collected to assess the activities of complexes I-III, II and IV of the respiratory chain, as well as ROS levels. The activities of complexes I-III, II and IV of the respiratory chain in gills decreased, while the ROS levels increased in infected compared to uninfected animals. Moreover, a significant negative correlation was found between enzymatic activity of the complexes I-III and IV related to ROS levels in P. aeruginosa infected animals, corroborating to our hypothesis that inhibition on complexes of respiratory chain leads to ROS formation. Also, microscopic severe gill damage and destruction of primary and secondary lamellae were observed in infected animals, with the presence of hyperplasia, leukocytic infiltration and telangiectasia. In summary, we have demonstrated, for the first time, that experimental infection by P. aeruginosa inhibits the activities of mitochondrial complexes of respiratory chain and, consequently, impairs the cellular energy homeostasis. Moreover, the inhibition on mitochondrial complexes I-III and IV are linked to the ROS production, contributing to disease pathogenesis.

Pseudomonas aeruginosa strain PA01 impairs enzymes of the phosphotransfer network in the gills of Rhamdia quelen.

Integration of mitochondria with cytosolic ATP-consuming/ATP-sensing and substrate supply processes is critical for gills bioenergetics, since this tissue plays an important role in the respiratory energy metabolism. The effects of bacterial infection on gills remain poorly understood, limited only to histopathological analyses. Thus, the aim of this study was to investigate whether experimental infection by Pseudomonas aeruginosa strain PA01 alters the enzymes of the phosphoryltransfer network (adenylate kinase (AK), pyruvate kinase (PK) and cytosolic and mitochondrial creatine kinase (CK)) in gills of silver catfish (Rhamdia quelen). The animals were divided into two groups with six fish each: uninfected (negative control) and infected (positive control). On day 7 post-infection (PI), animals were euthanized and the gills collected. AK, PK, and cytosolic and mitochondrial CK activities in gills decreased in infected compared to uninfected animals. Also, severe gill damage and destruction in the primary and secondary lamellae was observed in the infected animals. Therefore, we have demonstrated, for the first time, that experimental infection by P. aeruginosa inhibits key enzymes linked to the production and utilization of metabolic energy in silver catfish, and consequently, impairs cellular energy homeostasis, which may contribute to disease pathogenesis.

Nerolidol-loaded nanospheres prevent hepatic oxidative stress of mice infected by Trypanosoma evansi.

The aim of this study was to evaluate the effect of nerolidol free (N-F) and nerolidol-loaded in nanospheres (N-NS) on the hepatic antioxidant/oxidant status of mice experimentally infected by Trypanosoma evansi. In the liver it was measured: reactive oxygen species (ROS), thiobarbituric reactive acid substances (TBARS) and non-protein thiols (NPSH), catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) and performed histopathological examination. In addition, seric levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Liver samples from mice infected by T. evansi showed increased (P < 0·05) ROS, TBARS, AST and ALT levels and SOD activity, and decreased NPSH levels and CAT activity (P < 0·05) compared with uninfected animals. N-NS treatment prevented (P < 0·05) ROS and TBARS increase, and increased NPSH levels, and ameliorate CAT and SOD activities on liver of infected mice. Moreover, N-NS treatment reduced (P < 0·05) AST and ALT levels, and prevented histopathological changes caused by the parasite. N-NS protected the liver from the oxidative stress caused by T. evansi, which might be due to its antioxidant properties. Nerolidol might be considered a promising therapeutic agent against oxidative stress, and nanotechnology is an encouraging approach to be explored.

Nerolidol-loaded nanospheres prevent behavioral impairment via ameliorating Na+, K+-ATPase and AChE activities as well as reducing oxidative stress in the brain of Trypanosoma evansi-infected mice.

The aim of this study was to investigate the effect of nerolidol-loaded nanospheres (N-NS) on the treatment of memory impairment caused by Trypanosoma evansi in mice, as well as oxidative stress, and Na+, K+-ATPase and acetylcholinesterase (AChE) activities in brain tissue. Animals were submitted to behavioral tasks (inhibitory avoidance task and open-field test) 4 days postinfection (PI). Reactive oxygen species (ROS) and thiobarbituric acid-reactive substance (TBARS) levels and catalase (CAT), superoxide dismutase (SOD), Na+, K+-ATPase and AChE activities were measured on the fifth-day PI. T. evansi-infected mice showed memory deficit, increased ROS and TBARS levels and SOD and AChE activities, and decreased CAT and Na+, K+-ATPase activities compared to uninfected mice. N-NS prevented memory impairment and oxidative stress parameters (except SOD activity), while free nerolidol (N-F) restored only CAT activity. Also, N-NS treatment was able to prevent alterations in Na+, K+-ATPase and AChE activities caused by T. evansi infection. A significantly negative correlation was observed between memory and ROS production (p < 0.001; r = -0.941), as well as between memory and AChE activity (p < 0.05; r = -0.774). On the contrary, a significantly positive correlation between memory and Na+, K+-ATPase activity was observed (p < 0.01; r = 0.844). In conclusion, N-NS was able to reverse memory impairment and to prevent increased ROS and TBARS levels due to amelioration of Na+, K+-ATPase and AChE activities and to activation of the antioxidant enzymes, respectively. These results suggest that N-NS treatment may be a useful strategy to treat memory dysfunction and oxidative stress caused by T. evansi infection.

A novel approach to arthritis treatment based on resveratrol and curcumin co-encapsulated in lipid-core nanocapsules: In vivo studies.

Resveratrol and curcumin are two natural polyphenols extensively used due to their remarkable anti-inflammatory activity. The present work presents an inedited study of the in vivo antioedematogenic activity of these polyphenols co-encapsulated in lipid-core nanocapsules on Complete Freund's adjuvant (CFA)-induced arthritis in rats. Lipid-core nanocapsules were prepared by interfacial deposition of preformed polymer. Animals received a single subplantar injection of CFA in the right paw. Fourteen days after arthritis induction, they were treated with resveratrol, curcumin, or both in solution or loaded in lipid-core nanocapsules (1.75 mg/kg/twice daily, i.p.), for 8 days. At the doses used, the polyphenols in solution were not able to decrease paw oedema. However, nanoencapsulation improved the antioedematogenic activity of polyphenols at the same doses. In addition, the treatment with co-encapsulated polyphenols showed the most pronounced effects, where an inhibition of 37-55% was observed between day 16 and 22 after arthritis induction. This treatment minimized most of the histological changes observed, like fibrosis in synovial tissue, cartilage and bone loss. In addition, unlike conventionally arthritis treatment, resveratrol and curcumin co-encapsulated in lipid-core nanocapsules did not alter important hepatic biochemical markers (ALP, AST, and ALT). In conclusion, the strategy of co-encapsulating resveratrol and curcumin in lipid-core nanocapsules improves their efficacy as oedematogenic agents, with no evidence of hepatotoxic effects. This is a promising strategy for the development of new schemes for treatment of chronic inflammation diseases, like arthritis.