Viviane do Carmo Gonçalves Souza

Activity of the enzyme adenosine deaminase in serum, erythrocytes and lymphocytes of rats infected with Trypanosoma evansi.

In Trypanosoma evansi infections changes in the haemogram are commonly observed, and the enzyme adenosine deaminase (ADA) plays an important role in the production and differentiation of blood cells. Thus, the aim of this study was to evaluate the activity of ADA in serum, erythrocytes and lymphocytes of rats infected with T. evansi compared to non-infected rats. Thirty adult rats were used, divided into 3 uniform groups. The animals in groups A and B were infected intraperitoneally with 2 x 10⁶ trypomastigotes/rat. Rodents from group C (control group), were not-infected. Blood collection was performed on days 4 and 20 post-infection (p.i.) in order to obtain acute and chronic infection stages of disease. The blood was used to assess the activity of ADA. In the blood, reduced haematocrit and increased lymphocytes were correlated with ADA activity in erythrocytes and lymphocytes. We observed reduction of ADA activity in serum and erythrocytes in rats infected with T. evansi compared to non-infected rats (P < 0.05). ADA activity in lymphocytes was decreased after 4 days, when the parasitaemia was high and increased after 20 days, when the number of circulating parasites was low. In conclusion, our results showed that the ADA activity was altered in serum, lymphocytes and erythrocytes of rats, concomitantly with haematological parameters, in experimental infection by T. evansi.

Free and nanoencapsulated curcumin prevents cigarette smoke-induced cognitive impairment and redox imbalance.

Cigarette smoke-exposure promotes neurobiological changes associated with neurocognitive abnormalities. Curcumin, a natural polyphenol, have shown to be able to prevent cigarette smoke-induced cognitive impairment. Here, we investigated possible mechanisms involved in curcumin protection against cigarette smoke-induced cognitive impairment and, due to its poor bioavailability, we investigated the potential of using curcumin-loaded lipid-core nanocapsules (C-LNC) suspension. Rats were treated with curcumin and cigarette smoke, once a day, 5 days each week, for 30 days. Animals were divided into ten groups: I, control (vehicle/corn oil); II, curcumin 12.5mg/kg; III, curcumin 25mg/kg; IV, curcumin 50mg/kg; V, C-LNC 4 mg/kg; VI, tobacco exposed; VII, curcumin 12.5mg/kg along with tobacco exposure; VIII, curcumin 25mg/kg along with tobacco exposure; IX, curcumin 50mg/kg along with tobacco exposure; X, C-LNC 4 mg/kg along with tobacco exposure. Cigarette smoke-exposure impaired object recognition memory (P<0.001), indicated by the low recognition index, increased biomarkers of oxidative/nitrosative stress such as TBARS (P<0.05) and NOx (P<0.01), decreased antioxidant defenses such as NPSH content (P<0.01) and SOD activity (P<0.01) and inhibited the activities of enzymes involved in ion homeostasis such as Na(+),K(+)-ATPase and Ca(2+)-ATPase. Both curcumin formulations (free and nanoencapsulated) prevented the memory impairment, the redox imbalance and the alterations observed in the ATPases activities. Maintenance of ion homeostasis and redox balance is involved in the protective mechanism of curcumin against tobacco-induced cognitive impairment. Our results suggest that curcumin is a potential therapeutic agent for neurocognition and that C-LNC may be an alternative to its poor bioavailability.

Adenosine deaminase activity in serum, erythrocytes and lymphocytes of rats infected with Leptospira icterohaemorrhagiae.

Leptospirosis is a systemic disease of humans and domestic animals, mainly dogs, cattle and swine. The course of human leptospirosis varies from mild to severe fatal forms and the most severe form of human leptospirosis is principally caused by Leptospira interrogans serovar icterohaemorrhagiae (L. icterohaemorrhagiae). The enzyme adenosine deaminase (ADA) plays an important role in the production and differentiation of blood cells. The aim of this study was to evaluate the activity of ADA in serum, erythrocytes and lymphocytes of rats infected with L. icterohaemorrhagiae, as compared with non-infected rats. Twenty-four adult rats, divided into two uniform groups (A and B) were used for the enzymatic assays. The animals in Group B were inoculated intraperitoneally with 2×10(8) leptospires/rat, and the rodents in Group A (control) were not-inoculated. Blood collection was performed on days 5 and 15 post-infection (PI) and the blood used to assess the ADA activity. The infection by L.icterohaemorrhagiae altered erythrocyte count, hemoglobin concentration and hematocrit, causing a decrease in all these parameters on day 15 PI. Lymphocytes decreased significantly on day 15 PI, and ADA activity in serum was inhibited in infected rats on days 5 and 15 PI and its activity in erythrocytes were increased on day 5 PI. On day 5 PI, we found an increase in ADA activity in erythrocytes of infected rats. No correlation was observed between hematocrit and erythrocyte ADA activity on days 5 and 15 PI. The ADA activity was inhibited in rats infected on day 15 PI. A positive correlation (r(2)=60) was also observed between the number of lymphocytes and ADA activity in lymphocytes on day 15 PI (P<0.05). In conclusion, our results showed that the ADA activity is altered in serum, lymphocytes and erythrocytes in experimental infection by L.icterohaemorrhagiae in rats, concomitantly with hematological parameters.

Activities of enzymes that hydrolyze adenine nucleotides in lymphocytes from patients with rheumatoid arthritis.

The purpose of this study was to investigate the activities of ecto‐nucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5; CD39) and adenosine deaminase (E‐ADA; EC 3.5.4.4) in lymphocytes from patients with rheumatoid arthritis (RA). Thirty patients diagnosed with RA through American College of Rheumatology criteria as well as 30 healthy patients were selected. Peripheral blood lymphocytes were isolated, and E‐NTPDase and E‐ADA activities were assayed. The results demonstrated an increased E‐NTPDase activity (both ATP and ADP as substrates) and a decreased E‐ADA activity in RA patients. These data suggest an organic effort to preserve the adenosine level, which is known to have anti‐inflammatory and analgesic properties, working as a potent suppressor of immune response. Copyright

NTPDase activity in lymphocytes of rats infected by Trypanosoma evansi.

Trypanosoma evansi is the aetiological agent of trypanosomosis in domestic animals. In this pathology, an inflammatory response can be observed and, as a consequence, the increase of extracellular adenine nucleotides such as ATP. These nucleotide concentrations are regulated by ectoenzymes such as NTPDase (EC 3.6.1.5, CD39), which catalyses the hydrolysis of ATP and ADP into AMP. In this study, the activity of NTPDase in lymphocytes of rats experimentally infected with T. evansi was evaluated. The animals were inoculated with the parasite and monitored by blood smear on a daily basis. The animals were then were divided into 4 groups according to the degree of parasitaemia and period of infection. The blood collections for enzyme analysis and lymphocyte count were performed on the 3rd (beginning of infection), 5th (acute infection) and 15th (chronic infection) days post-infection (p.i.). The control group was composed of non-infected animals. In the infected group a decrease in ATP hydrolysis (36%) was observed on the 3rd day p.i. and a decrease in ADP hydrolysis (62%) was observed on the 5th day p.i. when compared to the control. On the 15th day p.i., an increase in ATP (94%) and ADP (50%) hydrolysis was observed in the infected group. Considering these data it is suggested that NTPDase activity is altered on the surface of lymphocytes of rats infected with T. evansi at different time-points of infection.

Immunotherapy for pythiosis: Effect on NTPDase activity in lymphocytes of an experimental model.

NTPDase (EC 3.6.1.5) occurs in lymphocytes and plays an important role in immune function, in that hydrolyzes extracellular nucleoside tri- and/or diphosphates to form AMP. Pythium insidiosum causes the disease pythiosis, a pyogranulomatous disease of horses, dogs, cattle, cats and humans. Most antifungal drugs are ineffective against this pathogen, and immunotherapy, a treatment approach that relies on the injection of P. insidiosum antigen, has been successfully used in humans and horses to manage this disease. In this study, we investigated NTPDase activity in lymphocytes from rabbits inoculated with zoospores of P. insidiosum. After immunotherapy, we investigated the relationship between enzymatic activity and the pattern of the immune response. One milliliter of zoospores was inoculated subcutaneously into the coastal region of each rabbit. An average of 17,500 viable mobile zoospores/mL of induction medium was administered. Inoculated rabbits were checked weekly, and the subcutaneous nodular area (cm²) was measured 28 days after inoculation. Rabbits that developed lesions received four doses of immunotherapy at intervals of 14 days. Blood samples were collected by heart puncture twice a month for the determination of NTPDase activity. The results demonstrated that NTPDase activity in lymphocytes was increased in relation to ATP hydrolysis (by about 100%) in pythiosis and returned to normal values after immunotherapy. The data demonstrating NTPDase activity before and after immunotherapy reinforce the previously elaborated hypothesis that the change from a Th2 to a Th1 immune response is responsible for the curative properties of immunotherapy.

E‐ADA activity in lymphocytes of an experimental model of pythiosis treated with immunotherapy

Pythiosis is a life‐threatening disease caused by the oomycete Pythium insidiosum. Some authors have suggested the involvement of a Th2‐like immune response in the infected host, which leads to extensive tissue damage. The switch from a Th2 to a Th1 response pattern is one hypothesis to explain the curative properties of immunotherapy. Taking into account the importance of immunotherapy for pythiosis treatment and the contribution of adenine nucleotides in the immunoregulation of the host, we evaluated the ecto‐adenosine deaminase (E‐ADA; EC 3·5.4·4) activity in lymphocytes from rabbits inoculated with P. insidiosum. Rabbits were inoculated with 1 milliliter of zoospores subcutaneously injected into the lateral thorax; after developing lesions, the rabbits received eight doses of immunotherapy. E‐ADA activity was measured in lymphocytes and the adenine nucleotides and adenosine levels were quantitatively determined in serum. Rabbits with characteristic lesions of pythiosis showed a decreased E‐ADA activity (82·36%), a decreased adenosine triphosphate concentration (54·04%) and a higher adenosine concentration (2·51 fold), when compared with controls, after 28 days of inoculation. However, after the immunotherapy, the rabbits showed an increase in the E‐ADA activity when compared with control (78·62%), contributing for the change in the immune response. Our results reinforce the hypothesis that the change from a Th2 to a Th1 immune response with the participation of the purinergic system could be responsible for the curative properties of immunotherapy. Copyright

Increased NTPDase activity in lymphocytes during experimental sepsis.

We investigated in rats induced to sepsis the activity of ectonucleoside triphosphate diphosphohydrolase (NTPDase; CD39; E.C. 3.6.1.5), an enzyme involved in the modulation of immune responses. After 12 hours of surgery, lymphocytes were isolated from blood and NTPDase activity was determined. It was also performed the histology of kidney, liver, and lung. The results demonstrated an increase in the hydrolysis of adenosine-5′-triphosphate (ATP) (P < 0.01), but no changes regarding adenosine-5′-monophosphate (ADP) hydrolysis (P > 0.05). Histological analysis showed several morphological changes in the septic group, such as vascular congestion, necrosis, and infiltration of mononuclear cells. It is known that the intracellular milieu contains much more ATP nucleotides than the extracellular. In this context, the increased ATPasic activity was probably induced as a dynamic response to clean up the elevated ATP levels resulting from cellular death.

Nicotine alters the ectonucleotidases activities in lymphocytes: In vitro and in vivo studies

The aim of the present study was to investigate the effects in vivo and in vitro of nicotine, an important immunosuppressive agent, on NTPDase and ADA activities in lymphocytes of adult rats. The following nicotine doses in vivo study were evaluated: 0.0, 0.25 and 1.0mg/kg/day injected subcutaneously in rats for 10days. The activity of the enzymes were significantly decreased with nicotine 0.25 and 1mg/kg which inhibited ATP (22%, 54%), ADP (44%, 30%) hydrolysis and adenosine (43%, 34%) deamination, respectively. The expression of the protein NTPDase in rat lymphocytes was decreased to nicotine 1mg/kg and the lymphocytes count was decreased in both nicotine doses studied. The purine levels measured in serum of the rats treated with nicotine 0.25mg/kg significantly increased to ATP (39%), ADP (39%) and adenosine (303%). The nicotine exposure marker was determinate by level of cotinine level which significantly increased in rats treated with nicotine 0.25 (39%) and 1mg/kg (131%) when compared to rats that received only saline. The second set of study was in vitro assay which the ATP-ADP-adenosine hydrolysis were decreased by nicotine concentrations 1mM (0% - 0% - 16%, respectively), 5mM (42% - 32% - 74%, respectively), 10mM (80% - 27% - 80%, respectively) and 50mM (96% - 49% - 98%, respectively) when compared with the control group. We suggest that alterations in the activities of these enzymes may contribute to the understanding of the mechanisms involved in the suppression of immune response caused by nicotine.

An experimental model of contact dermatitis: Evaluation of the oxidative profile of Wistar rats treated with free and nanoencapsulated clobetasol

Abstract Objective An experimental animal model of contact dermatitis (CD) was used to investigate the effects of free and nanoencapsulated clobetasol propionate on the skin and on the oxidative profile of liver tissue. Methods Female Wistar rats were divided into six groups, each containing eight rats. The first group, control (C), was sensitized with solid vaseline. Group 2, (CD), was sensitized with 5% NiSO4. Groups 3 and 4 were sensitized with 5% NiSO4 and treated with free (FC) and nanoencapsulated (NC) clobetasol (0.42 mg/g), respectively, daily for 5 days. Group 5 was treated with nanoencapsulated clobetasol (0.42 mg/g) on days 1, 3, and 5 (C135) and group 6 received a hydrogel containing empty nanoparticles (NP) daily for 5 days. Thiobarbituric acid reactive substances (TBARS), carbonyl levels, non-protein sulfhydryl groups (NPSH) and catalase activity were measured in liver homogenates. Results A significant increase was observed in the levels of TBARS, NPSH, and catalase activity for the groups CD and NP. Discussion Our results suggest that both NiSO4 sensitization and NP administration induced oxidation of cellular lipids and activated the antioxidant enzyme catalase to protect from this damage. These results also indicated that daily treatment with the free and nanoencapsulated clobetasol, as well as treatment with the nanoencapsulated clobetasol every other day, were able to prevent these redox alterations and protect against histological damage.