Nathieli B. Bottari

Activity of cholinesterases, pyruvate kinase and adenosine deaminase in rats experimentally infected by Fasciola hepatica: Influences of these enzymes on inflammatory response and pathological findings

The aim of this study was to investigate acetylcholinesterase (AChE) in total blood and liver tissue; butyrylcholinesterase (BChE) in serum and liver tissue; adenosine deaminase (ADA) in serum and liver tissue; and pyruvate kinase (PK) in liver tissue of rats experimentally infected by Fasciola hepatica. Animals were divided into two groups with 12 animals each, as follows: group A (uninfected) and group B (infected). Samples were collected at 20 (A1 and B1;n=6 each) and 150 (A2 and B2; n=6 each) days post-infection (PI). Infected animals showed an increase in AChE activity in whole blood and a decrease in AChE activity in liver homogenates (P<0.05) at 20 and 150 days PI. BChE and PK activities were decreased (P<0.05) in serum and liver homogenates of infected animals at 150 days PI. ADA activity was decreased in serum at 20 and 150 days PI, while in liver homogenates it was only decreased at 150 days PI (P<0.05). Aspartate aminotransferase and alanine aminotransferase activities in serum were increased (P<0.05), while concentrations of total protein and albumin were decreased (P<0.05) when compared to control. The histological analysis revealed fibrous perihepatitis and necrosis. Therefore, we conclude that the liver fluke is associated with cholinergic and purinergic dysfunctions, which in turn may influence the pathogenesis of the disease.

Novel markers of inflammatory response and hepatic dysfunction in canine leishmaniasis.

Dogs are the main host of Leishmania infantum, and the clinical presentation may range from asymptomatic to systemic manifestations. The immune mechanisms in infected, but clinically healthy dogs, prevails Th1 response mediated by cytokines. In this sense, adenosine deaminase (ADA) and butyrylcholinesterase (BChE) are considered as key enzymes in several physiological processes, including the modulation of inflammatory process. Considering the variable immune response against Leishmania and the known participation of ADA and BChE, the aim of this study was to assess the relation between these two enzymes with the inflammatory response as well as hepatic function in dogs naturally infected with L. infantum. For this purpose, the activity of ADA and BChE was assessed in sera of 24 dogs naturally infected with L. infantum, plus 17 healthy dogs. The naturally infected dogs had clinical signs compatible with leishmaniasis and sera activities of ADA (P<0.01) and BChE (P<0.05) decreased, when compared to the healthy group. The reduction of ADA activity probably represented an effect on inflammatory response, especially due to the decreased hydrolysis of extracellular adenosine, might in order to protect against tissue damage and, also, setting a down-regulation on pro-inflammatory cytokines. BChE enzyme had no effect on modulating the immune response in leishmaniasis, but it decreased, a fact may related to deficiency of synthesis in the liver. Therefore, ADA and BChE activities reduced probably in order to protect against extra tissue damage and due failure in synthesis, respectively.

Serum levels of LH, FSH, estradiol and progesterone in female rats experimentally infected by Trypanosoma evansi.

The goal of this study was to evaluate reproductive hormones in sera samples of female rats experimentally infected by Trypanosoma evansi during different phases of the estrous cycle. For that, 64 animals were divided into two groups: 24 rats for the control group (uninfected), and 40 animals were infected by T. evansi. These groups were divided into subgroups according to the time of infection (days 5 and 15 post-infection; PI) and the phase of the estrous cycle (proestrus, estrus, metestrus and diestrus). Serum was collected at days 5 and 15 PI and the levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone and estradiol were assessed by enzyme immunoassay technique. The concentration of nitrite/nitrate (NOx), advanced oxidation protein products (AOPP), and thiobarbituric acid reactive substances (TBARS) were measured in ovaries and uteruses in these same periods. Infected females showed significant decrease (P<0.05) of LH, FSH, estradiol and progesterone in different periods and phases of the estrous cycle when compared to uninfected rats. In addition, it was observed an increase in the concentration of NOx, AOPP, and TBARS in the ovaries, which is indicative of cell damage. Therefore, our experimental study showed that T. evansi infection in female rats may cause changes in LH, FSH, estradiol, and progesterone levels regardless of the time of infection or phase of the estrous cycle.

Fowl typhoid in laying hens cause hepatic oxidative stress

The aim of this study was to analyses nitric oxide, antioxidant status, and oxidative profile in the liver of laying hens naturally infected by Salmonella enterica subsp enterica serovar Gallinarum (S. Gallinarum). The nitrite/nitrate (NOx), reactive oxygen species (ROS), thiobarbituric acid-reactive substances (TBARS), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities were measured in liver samples, as well the biomarkers of hepatic function (alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), total protein and albumin levels measured in serum. NOx levels and CAT activity were reduced in hepatic tissue of infected hens. On the other hand, TBARS and ROS levels, GR, GPx and GST activities were higher in infected animals. On biomarkers of tissue damage, ALT, AST, GGT and total protein levels were higher in serum of infected hens, and showed decreased albumin levels. In summary, ROS and TBARS production lead to damage on the membrane lipids that alter activities of antioxidant enzymes CAT, GR, GPx and GSH, an adaptive response against S. Gallinarum infection, contributing to the pathophysiology and clinical signs of the disease.

Butyrylcholinesterase as a marker of inflammation and liver injury in the acute and subclinical phases of canine ehrlichiosis.

The aim of this study was to evaluate the role of butyrylcholinesterase (BChE) as a marker of inflammation and liver injury in the acute and subclinical phases of canine ehrlichiosis. Forty-two serum samples of dogs naturally infected with Ehrlichia canis were used, of which 24 were from animals with the acute phase of the disease and 18 with subclinical disease. In addition, sera from 17 healthy dogs were used as negative controls. The hematocrit, BChE activity, hepatic injury (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)), nitric oxide, and cytokines levels were evaluated. The BChE activity was significantly elevated (P<0.05) in dogs with the acute phase of the disease when compared to healthy animals. However, there was a reduction on BChE activity on dogs with subclinical disease compared to the other two groups. AST and ALT levels were significantly higher (P<0.05) in the acute phase, as well as the inflammatory mediators (NOx, TNF-α, INF-γ, IL-4, IL-6) when compared to the control group. On the other hand, IL-10 levels were lower in the acute phase. Based on these results, we are able to conclude that the acute infection caused by E. canis in dogs leads to an increase on seric BChE activity and some inflammatory mediators. Therefore, this enzyme might be used as a marker of acute inflammatory response in dogs naturally infected by this bacterium.

Effects of sulfamethoxazole-trimethoprim associated to resveratrol on its free form and complexed with 2-hydroxypropyl-β-cyclodextrin on cytokines levels of mice infected by Toxoplasma gondii

The aim of this study was to investigate the effects of resveratrol on its free form and complexed with 2-hydroxypropyl-β-cyclodextrin (HPβCD) when associated with sulfamethoxazole-trimethoprim (ST) on cytokines levels of mice (n = 60) experimentally infected by Toxoplasma gondii. Groups A and E were used as controls (untreated): negative and positive, respectively. The onset of treatment started 20 days post-infection (PI), and it lasted for 10 consecutive days. ST was administered orally in doses of 0.5 mg kg(-1) for groups B and F, while 100 mg kg(-1) was the dose for resveratrol in its free form (groups C - G), inclusion complex (groups D and H), and on free and inclusion complex together (groups I - J). On day 31 PI, blood samples were collected in order to evaluate the cytokine profile. The mice that received drug combination (I and J) showed a significant (P < 0.05) reduction in the number of cysts in the brain compared to other infected groups (E - H). The results showed that mice from the Group E had increased (P < 0.001) levels of pro-inflammatory cytokines, while IL-10 levels were reduced when compared to the Group A. Additionally, there were increased levels of IL-4 and IFN-γ in animals of groups C and D, respectively (P < 0.05). Animals of the Group B showed reduced levels of IL-1, IL-4, IL-6, TNF-α, and IFN-γ (P < 0.05). Mice infected and treated (groups F - J) showed increased levels of pro-inflammatory cytokines along with a reduction of IL-10. Treatment with the combination of drugs (the Group J) led to a protective effect, i.e. reduction in pro-inflammatory cytokines. Therefore, resveratrol associated with ST was able to modulate seric cytokine profile and moderate the tissue inflammatory process caused by T. gondii infection, as well as to reduce parasite multiplication.

Dietary Supplementation of Ginger and Turmeric Rhizomes Modulates Platelets Ectonucleotidase and Adenosine Deaminase Activities in Normotensive and Hypertensive Rats.

Hypertension is associated with platelet alterations that could contribute to the development of cardiovascular complications. Several studies have reported antiplatelet aggregation properties of ginger (Zingiber officinale) and turmeric (Curcuma longa) with limited scientific basis. Hence, this study assessed the effect of dietary supplementation of these rhizomes on platelet ectonucleotidase and adenosine deaminase (ADA) activities in Nω‐nitro‐l‐arginine methyl ester hydrochloride (l‐NAME) induced hypertensive rats. Animals were divided into seven groups (n = 10): normotensive control rats; induced (l‐NAME hypertensive) rats; hypertensive rats treated with atenolol (10 mg/kg/day); normotensive and hypertensive rats treated with 4% supplementation of turmeric or ginger, respectively. After 14 days of pre‐treatment, the animals were induced with hypertension by oral administration of l‐NAME (40 mg/kg/day). The results revealed a significant (p < 0.05) increase in platelet ADA activity and ATP hydrolysis with a concomitant decrease in ADP and AMP hydrolysis of l‐NAME hypertensive rats when compared with the control. However, dietary supplementation with turmeric or ginger efficiently prevented these alterations by modulating the hydrolysis of ATP, ADP and AMP with a concomitant decrease in ADA activity. Thus, these activities could suggest some possible mechanism of the rhizomes against hypertension‐derived complications associated to platelet hyperactivity. Copyright

Sulfamethoxazole-trimethoprim associated with resveratrol for the treatment of toxoplasmosis in mice: Influence on the activity of enzymes involved in brain neurotransmission.

This study aimed to investigate the influence of sulfamethoxazole-trimethoprim (ST) associated with resveratrol on the enzymatic activities of acetylcholinesterase (AChE), adenylate kinase (AK), pyruvate kinase (PK), and creatine kinase (CK) in the brain of mice experimentally infected by Toxoplasma gondii. For that, 60 mice were divided into ten groups with 6 animals each: groups A to D composed by healthy mice and groups E to J consisting of animals infected by T. gondii (VEG strain). Animals started treatment 20 days post-infection for 10 consecutive days with oral doses of 0.5 mg kg(-1) of ST (groups B and F), 100 mg kg(-1) of free resveratrol (groups C and G) and inclusion complex of resveratrol (nanoparticles containing resveratrol) (groups D and H), as well as with an association of both drugs (groups I and J). The results showed increased (P < 0.001) AChE activity on infected animals (groups E-J) when compared to not-infected (A) animals, and also uninfected animals treated with ST (group B) had increased AChE activity. AK activity decreased (P < 0.001) in the infected and untreated (group E), differently from the other groups that did not differ. PK activity did not differ between groups (P > 0.05). When comparing control groups (uninfected (A) and infected (E)), we verified a significant (P < 0.001) increase in CK activity in the brain, and it is noteworthy that the animals treated with resveratrol associated with ST (group I and J) had similar CK activity to those animals from the group A. Treatment with the combination of ST and resveratrol was able to reduce (P < 0.05) the number of parasitic cysts in the brain, thus reduced inflammatory infiltrates in the liver, and prevented the occurrence of hepatocytes lesions due to toxoplasmosis in mice. Based on these results, it is possible to conclude that increased AChE and CK activities after T. gondii infection did not change with the treatment of ST-resveratrol association. In addition, decreased AK activity caused by T. gondii infection was normalized by ST-resveratrol treatment. T. gondii infection and treatment does not affect PK activity in brain.

Cattle naturally infected by Eurytrema coelomaticum: Relation between adenosine deaminase activity and zinc levels

The enzyme adenosine deaminase (ADA) is critical for modulating the immune system, and in the presence of zinc, its activity is catalyzed. The aim of this study was to evaluate the ADA activity in pancreas of cattle naturally infected by Eurytrema coelomaticum in relation to the results of zinc levels, pathological findings and parasite load. For this study 51 slaughtered cattle were used. The animals were divided into two groups: Group A consisting of animals naturally infected by E. coelomaticum (n=33) and Group B of uninfected animals (n=18). Blood and pancreas were collected of each animal for analysis of zinc and ADA, respectively. Infected cattle showed a reduction on seric levels of zinc, and decreased ADA activity in the pancreas (P>0.05). A positive correlation between zinc levels and ADA activity was observed. Thus, high parasite load and severity of histopathologic lesions affect the ADA activity in pancreas, as well as the zinc levels in serum of infected animals (negative correlation between these variables). Therefore, we can conclude that cattle infected by E. coelomaticum have low ADA activity in pancreas, which can be directly related to zinc reduction, responsible for ADA activation and catalyzes.

Oxidative stress and inflammatory response biomarkers in dogs with mammary carcinoma

Mammary carcinoma is the most common cancer that affects dogs, and in many cases it leads to death. Thus, given the importance of this disease, to clarify its pathogenesis is an important measure. In this sense, the aim of this study was to investigate the levels of cytokines and nitric oxide (NO), oxidative and antioxidant status, as well as the activity of adenosine deaminase (ADA) and butyrylcholinesterase (BChE) in dogs diagnosed with mammary carcinoma. With this purpose, thirty-three (33) serum samples from female dogs with histopathological diagnosis of mammary carcinoma, without evidence of metastasis, were used (group B). The material was classified based on the degree of malignancy, as follows: subgroup B1 (low-grade malignancy; n=26) and subgroup B2 (high grade of malignancy; n=7). Serum samples from healthy females (group A; n=10) were used as negative control. Our results showed that levels of cytokines (TNF-α, INF-γ, IL-1, and IL-6), NOx (nitrite/nitrate), AOPP (protein oxidation), and FRAP (antioxidant power) were significantly (P<0.05) increased in dogs with mammary carcinoma (group B), when compared with group A. On the other hand, ADA activity was significantly decreased (P<0.05) in both subgroups B1 and B2, when compared with group A. BChE activity, however, was reduced (P<0.05) only in subgroup B2 when compared with group A and subgroup B1. Unlike other variables, NO, AOPP, and IFN-γ were influenced by the degree of tumor malignancy, i.e., their levels were even higher in subgroup B2. Therefore, based on these results, we can conclude that all variables investigated are related to the pathogenesis of this disease, since they were altered in dogs with mammary tumor. Additionally, we suggest that ADA activity had an anti-inflammatory effect on these tumor samples, probably in order to modulate the inflammatory response.