Distinct kinetics for nucleotide hydrolysis in lymphocytes isolated from blood, spleen and cervical lymph nodes: Characterization of ectonucleotidase activity.

Ectonucleotidases are a plasma membrane-bound enzyme that hydrolyses extracellular adenosine triphosphate (eATP) and adenosine diphosphate (eADP) to adenosine monophosphate (AMP). It regulates normal function of lymphocytes, acts as an inflammatory marker and represents a molecular target for new therapeutics. Thus, this study sought to isolate lymphocytes from blood (BL), spleen (SL) and cervical lymph node (CLL), and characterize the eATP and eADP enzymatic hydrolysis in Wistar rats. The hydrolysis of the nucleotides occurred primarily at pH 8.0, 37°C in the presence of Ca2+ or Mg2+ . Chevillard-plot showed the hydrolysis of eATP and eADP at the same active site. The inhibitors of some classical ATDPases did not cause any significant change on enzymatic activity. Inhibitors of E-NTPDase (-1, -2, -3 isoforms) and E-NPP-1 decrease the enzyme activity in all resident lymphocytes. Furthermore, kinetic parameters (Vmax and Km) revealed that SL had significantly (P < .001) higher enzymatic activity when compared to BL and CLL. In conclusion, this study standardized kinetic values for eATP and eADP hydrolysis for resident lymphocytes isolated from BL, SL and CLL.

Lipotoxicity-associated inflammation is prevented by guarana (Paullinia cupana) in a model of hyperlipidemia.

Hyperlipidemia causes lipotoxicity which prompts an inflammatory response linked to the development of cardiovascular diseases. Natural compounds have been receiving special attention for its potential to treat diseases, inexpensiveness, and safety. Guarana (Paullinia cupana) has demonstrated notable anti-inflammatory and antioxidant effects, which may prevent chronic diseases caused by changes in lipid profile. Thus, this study aims to evaluate the effect of guarana powder (Paullinia cupana) in the purine metabolism and inflammatory profile in lymphocytes and serum of rats with Poloxamer-407-induced hyperlipidemia. Pretreatment with guarana 12.5, 25, and 50 mg/kg/day or caffeine (0.2 mg/kg/day) by gavage was applied to adult male Wistar rats for a period of 30 days. As a comparative standard, we used simvastatin (0.04 mg/kg) post-induction. Hyperlipidemia was acutely induced with intraperitoneally injection of Poloxamer-407 (500 mg/kg). Guarana powder and caffeine increased the activity of the E-NTPDase (ecto-apyrase), and all pretreatments decreased the E-ADA (ecto-adenosine deaminase) activity, reducing the inflammatory process caused by lipotoxicity. In hyperlipidemic rats, ATP levels were increased while adenosine levels were decreased, guarana and caffeine reverted these changes. Guarana powder, caffeine, and simvastatin also prevented the increase in INF-γ and potentiated the increase in IL-4 levels, promoting an anti-inflammatory profile. Guarana promoted a more robust effect than caffeine. Our results show that guarana powder and caffeine have an anti-inflammatory as seen by the shift from a proinflammatory to an anti-inflammatory profile. The effects of guarana were more pronounced, suggesting that guarana powder may be used as a complementary therapy to improve the lipotoxicity-associated inflammation.

CD4/CD8 ratio, comorbidities, and aging in treated HIV infected individuals on viral suppression.

The progression of the human immunodeficiency virus (HIV) infection to acquired immunodeficiency syndrome (AIDS) can be efficiently interrupted by antiretroviral therapy (ART). However, even successfully treated HIV-infected individuals are prone to develop non-AIDS-related diseases that affect the metabolism and several organs and systems. Biomarkers that predict the occurrence of comorbidities may help develop preventive measures. Current research shows that CD4+ T cell counts and viral load do not predict the development of non-AIDS-related diseases. The CD4/CD8 ratio has been indicated as a suitable marker of persistent immune dysfunction and the occurrence of non-AIDS-related events in treated HIV-positive patients. In this study, we explored the relationship between CD4/CD8 ratios, comorbidities, and aging in ART-treated HIV patients on viral suppression. We collected and evaluated data from 352 HIV-positive adults who were virologically suppressed (<40 copies/mL) on ART and with CD4 counts above 350 cells/mm3 . The median age for participants was 46 years, 218 individuals had at least one comorbidity, and 239 had inverted CD4/CD8 ratios (<1). Current CD4/CD8 ratios were predicted by baseline CD4/CD8 ratios and nadir CD4 counts. Despite the high rates of inverted CD4/CD8 ratios and prevalence of comorbidities, no association between them was observed. The prevalence of comorbidities was significantly higher in older individuals, though aging alone did not explain the rate of all individual comorbidities. Low CD4/CD8 ratios were linked to neurocognitive disorders, suggesting that persistent T cell dysfunction contributes to neurocognitive decline.

Rutin and curcumin reduce inflammation, triglyceride levels and ADA activity in serum and immune cells in a model of hyperlipidemia.

Hyperlipidemia is associated with endothelial dysfunction and inflammatory disorders. Adenosine and adenosine deaminase (ADA) modulate immune responses and lipid metabolism. Curcumin and rutin are polyphenols with antioxidant, anti-inflammatory, and hypolipidemic effects. We evaluated the action of rutin and curcumin in the lipid levels and inflammation, as well as their effect on ADA activity in serum, lymphocytes, platelets, and neutrophils of hyperlipidemic rats. Adult male Wistar rats pretreated with curcumin and/or rutin for 30 days were submitted to Poloxamer-407- induced hyperlipidemia. Biochemical, hematological, and oxidative stress parameters, as well as serum and extracellular ADA activity, were performed 36h post-induction. Hyperlipidemia was confirmed by the increase in total cholesterol (TC) and triglycerides (TG). Hematological alterations, elevated reactive oxygen species (ROS) levels, and increased myeloperoxidase (MPO) and ADA activities were observed in hyperlipidemic rats. Curcumin and the curcumin/rutin association decreased TG and increased high-density lipids (HDL) levels. The pretreatments prevented changes in the hematological parameters, decreased the activities of MPO in plasma and ADA in serum and cells. Cholesterol and ROS levels were not altered by the pretreatments. Our results show that pretreatments with rutin and/or curcumin prevent the hyperlipidemia-induced inflammation. Pretreatments with curcumin and/or rutin are potential complementary therapies in the prevention of hypertriglyceridemia and inflammation.

Changes in inflammatory/cardiac markers of HIV positive patients.

HIV replication promotes atherogenesis and participates in the immune response to the virus, thereby influencing the inflammatory profile. These changes may, in turn, contribute to the risk of cardiovascular diseases with involvement of platelets. However, adenine nucleotides and nucleosides involved in thromboregulation and modulation of immune response may therefore be affected by these alterations. OBJECTIVES: This study sought to evaluate the profile of pro and anti-inflammatory cytokines (IL-10, IL-6, IL-17, TNF, IL-4, IL-2 and IFN-gamma), cardiac markers (troponin, CK, CK MB, LDH, CRP) in HIV-positive patients and assess the in vitro effect of antiretroviral therapy on the activities of ectonucleotidases (E-NTPDase and E-5'-nucleotidase) in human platelets. DESIGN AND METHODS: Blood samples were obtained from ten HIV positive patients at the Infectious Disease Clinic of the University Hospital of Santa Maria, Brazil and ten HIV negative individuals (control group) for this study. RESULTS: The results revealed that there were significant (P < 0.05) increases in serum levels of IL-6 and IFN-gamma with no significant (P > 0.05) changes in the serum levels of the cardiac markers investigated (CK, CK-MB, troponin, LDH and CRP). In addition, the ectonucleotidases (E-NTPDase and E-5'-nucleotidase) activities were not altered (P > 0.05) in human platelets when incubated with different antiretroviral drugs in vitro. CONCLUSIONS: The results of this study suggest that, despite successful treatment, a proinflammatory state is not altered in HIV patients, and that antiretroviral therapy per se does not change the purinergic profile.

Pretreatment with quercetin prevents changes in lymphocytes E-NTPDase/E-ADA activities and cytokines secretion in hyperlipidemic rats.

Hyperlipidemia (HL) is a condition associated with endothelial dysfunction and inflammatory disorders. Purinergic system ectoenzymes play an important role in modulating the inflammatory and immune response. This study investigated whether the preventive treatment with quercetin is able to prevent changes caused by hyperlipidemia in the purinergic system, through the activities of E-NTPDase and E-ADA in lymphocytes, and quantify the nucleotides and nucleoside, and the secretion of anti- and proinflammatory cytokines. Animals were divided into saline/control, saline/quercetin 5 mg/kg, saline/quercetin 25 mg/kg, saline/quercetin 50 mg/kg, saline/simvastatin (0.04 mg/kg), hyperlipidemia, hyperlipidemia/quercetin 5 mg/kg, hyperlipidemia/quercetin 25 mg/kg, hyperlipidemia/quercetin 50 mg/kg, and hyperlipidemia/simvastatin. Animals were pretreated with quercetin for 30 days and hyperlipidemia was subsequently induced by intraperitoneal administration of 500 mg/kg of poloxamer-407. Simvastatin was administered after the induction of hyperlipidemia. Lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Serum was separated for the cytokines and nucleotide/nucleoside quantification. E-NTPDase and E-ADA activities were increased in lymphocytes from hyperlipidemic rats and pretreatment with quercetin was able to prevent the increase in the activities of these enzymes caused by hyperlipidemia. Hyperlipidemic rats when receiving pretreatment with quercetin and treatment with simvastatin showed decreased levels of ATP and ADP when compared to the untreated hyperlipidemic group. The IFN-γ and IL-4 cytokines were increased in the hyperlipidemic group when compared with control group, and decreased when hyperlipidemic rats received the pretreatment with quercetin. However, pretreatment with quercetin was able to prevent the alterations caused by hyperlipidemia probably by regulating the inflammatory process. We can suggest that the quercetin is a promising compound to be used as an adjuvant in the treatment of hyperlipidemia.

Ecto-enzymes activities in splenic lymphocytes of mice experimentally infected by Trypanosoma cruzi and treated with specific avian immunoglobulins: an attempt to improve the immune response.

The aim of this study was to evaluate the therapeutic efficacy of specific avian polyclonal antibodies (IgY) against Trypanosoma cruzi and their interaction with ecto-enzymes of the purinergic system (NTPDase and adenosine deaminase (ADA) activities) in splenic lymphocytes. For this, mice were divided into six groups: three non-infected (A, B, and C) and three infected (D, E, and F). The groups A and D were composed by negative and positive controls, respectively; while the groups B and E were treated prophylactically with IgY (50 mg/kg), and the groups C and F were treated therapeutically with IgY (50 mg/kg). Treatment with IgY reduced parasitemia on day 6 post-infection (PI) compared to the infected control group, but it was similar on day 8 PI. Moreover, infected and treated animals (the groups E and F) did not show neither amastigotes in the cardiac tissue nor cardiac lesions when compared to the positive control group (the group D). The E-NTPDase (ATP and ADP as substrate) and ADA activities in splenic lymphocytes increased significantly in the positive control group (the group D) compared to the negative control group (the group A). The therapeutic treatment of IgY (the group F) was able to prevent the increase of E-NTPDase and E-ADA activities compared to the positive control group (the group D), but this finding was not observed in animals that received the prophylactic treatment (the group E). The therapeutic treatment of IgY may be considered an interesting approach to improve the immune response of mice experimentally infected by T. cruzi.

Melaleuca alternifolia essential oil prevents alterations to purinergic enzymes and ameliorates the innate immune response in silver catfish infected with Aeromonas hydrophila.

Aeromonas hydrophila infection represents a major impediment to the development of aquaculture, leading to important economic losses. Over the last few years, different methods have been used to counteract and minimize the negative effects of this infection, such as the use of Melaleuca alternifolia essential oil, popularly known as tea tree oil (TTO), that possess a bactericide action against A. hydrophila. The purinergic system develops an important role in the inflammatory response, principally due to involvement of adenosine triphosphate (ATP) in the inflammatory process, as well as by the anti-inflammatory properties of adenosine (Ado), a molecule that is controlled by NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) enzymes. Thus, the aim of this study was to investigate the involvement of purinergic enzymes in the pathogenesis of A. hydrophila infection, and whether the purinergic pathway and innate immune response are involved in the protective effects of TTO in silver catfish (Rhamdia quelen) experimentally infected with A. hydrophila. Our results revealed that A. hydrophila infection increased seric NTPDase and 5'-nucleotidase activity, while ADA activity decreased. Also, the seric levels of pro-inflammatory cytokines such as interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha (TNF-α) and interferon gamma (INF-γ) increased in the infected fish, while the seric level of anti-inflammatory interleukin-10 (IL-10) decreased. Treatment with TTO was able to prevent the impairment of purinergic enzymes and improve the innate immune response through the modulation of cytokine response during A. hydrophila infection. In summary, prophylactic therapy with TTO can be considered an important approach to improve the immune response and consequently avoid the inflammatory process in fish infected with A. hydrophila.

Hepatic xanthine oxidase activity and purine nucleosides levels as physiological mediators to analyze a subcutaneous treatment with (PhSe)2 in mice infected by Toxoplasma gondii.

The aim of this study was to evaluate the levels of purine nucleosides and xanthine oxidase (XO) activity in the liver of mice chronically infected by Toxoplasma gondii and treated with diphenyl diselenide (PhSe)2. For this experiment, forty Swiss mice were used. Twenty animals were orally infected by approximately 50 bradizoites of a cystogenic ME-49 strain of T. gondii, and the same number of uninfected mice was used as a control group. Ten infected and ten uninfected mice were subcutaneously treated twice (days 1 and 20 post-infection (PI)) with 5 µmol kg-1 of (PhSe)2. On day 30 PI, liver samples were collected to measure the levels of hypoxanthine (HYPO), xanthine (XAN), uric acid (UA), and XO activity. Infected animals showed increased (P < 0.05) levels of hepatic XAN and UA, as well as XO activity compared to uninfected animals. The use of (PhSe)2 in healthy mice increased the levels of all nucleosides, but decreased XO activity compared to healthy untreated animals. The group of infected and treated animals showed increased XAN and UA levels, and XO activity compared to the healthy control group, however infected and treated mice showed a decrease in the XO activity compared to the infected untreated group. We conclude that chronic infection caused by T. gondii can induce hepatic changes, such as increased UA levels and XO activity, that can increase the pro-oxidative profile. The (PhSe)2 treatment of healthy animals altered the levels of nucleosides, possibly due to low XO activity that decreased nucleoside degradation. Finally, (PhSe)2 treatment decreased XO activity in the infected group and increased nucleoside levels; however it was unable to reduce the UA levels found during the infection.

Treatment with 3'-deoxyadenosine and deoxycoformycin in mice infected by Trypanosoma cruzi and its side effect on purinergic enzymes.

The aim of this study was to evaluate the efficacy of 3'-deoxyadenosine and deoxycoformycin combination in the treatment of mice infected by T. cruzi, as well as to verify the influence of the treatment on purinergic enzymes. Heart and serum samples were collected from 60 mice (30 infected and 30 uninfected) at day 12 post-infection. To verify treatment efficacy, parasitemia was monitored, and the treatment with 3'-deoxy adenosine and deoxycoformycin combination was able to reduce it, but had no curative effect on mice. Seric activities of NTPDase (ATP and ADP substrate) and ADA were increased significantly in untreated mice infected by T. cruzi compared to the negative control, as well as mice treated with 3'-deoxyadenosine and deoxycoformycin (alone or combined) modulated the activity of NTPDase (ATP and ADP substrate), preventing them from increasing in infected animals (activity similar to healthy animals). Treatment with deoxycoformycin alone and associated with 3'-deoxyadenosine modulated the activity of ADA preventing them from increasing in infected animals. However, seric activities of ADA in mice treated with 3'-deoxyadenosine (cordycepin) alone does not modify the ADA activity compared with infected and non-treated mice. However, the 5'-nucleotidase activity decreased significantly in infected untreated animals and the same occurred in infected and treated animals with deoxycoformycin and 3'-deoxyadenosine. However, treatment with deoxycoformycin associated with 3'-deoxyadenosine preventing them from decreasing the 5'-nucleotidase activity. Therefore, we conclude that the treatments did not have curative success for mice infected by T. cruzi. However, the treatments were able to modulate the purinergic enzymes during the infection by T. cruzi, which may contribute to reduce the inflammatory damage in heart.

Sepsis induced by cecal ligation and perforation (CLP) alters nucleotidase activities in platelets of rats.

Sepsis is a potentially lethal condition, and it is associated with platelet alterations. The present study sought to investigate the activity of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'-nucleotidase, and ecto-adenosine deaminase (E-ADA) in the platelets of rats that were induced with sepsis. Male Wistar rats were divided into three groups of ten animals each: a negative control group (normal; NC); a group that underwent surgical procedures (sham); and a group that underwent cecal ligation and perforation (CLP). The induction of sepsis was confirmed by bacteremia, and the causative pathogen identified was Escherichia coli. Hematological parameters showed leukocytosis and thrombocytopenia in animals in the septic group. The results also revealed that there were significant (p < 0.05) increases in adenosine triphosphate (ATP) and adenosine monophosphate (AMP) hydrolyses, and in the deamination of adenosine in the CLP group compared to the sham and control groups. Conversely, ADP hydrolysis was significantly decreased (p < 0.05) in the CLP group compared to the sham and control groups. Purine levels were analyzed by high-performance liquid chromatography (HPLC) in serum samples from control, sham, and CLP groups. Increased concentrations of ATP, adenosine, and inosine were found in the CLP group compared to the sham and control groups. Conversely, the concentrations of ADP and AMP in the CPL group were not significantly altered. We suggest that alterations in hematological parameters, nucleotide hydrolysis in platelets, and nucleotide concentrations in serum samples of rats with induced sepsis may be related to thromboembolic events.

Enzymes that hydrolyze adenine nucleotides in a model of hypercholesterolemia induced by Triton WR-1339: protective effects of ß-caryophyllene.

Purinergic system has been proven to play a critical role in the inflammatory process and to represent an important therapeutic target to improve the immune response during hypercholesterolemia. ß-caryophyllene, a phytocannabinoid compound, has a powerful hypolipidemic and anti-inflammatory actions. However, the effects of ß-caryophyllene on seric enzymes of purinergic system have not been evaluated. The purpose of this study was to investigate whether ß-caryophyllene is able to ameliorate the seric activities of NTPDase and adenosine deaminase (ADA) in a model of hypercholesterolemia induced by Triton WR-1339. The activities of NTPDase and ADA were evaluated enzymatically, and the seric levels of ß-caryophyllene were evaluated by high-performance liquid chromatography. We found that treatment with ß-caryophyllene ameliorates the enzymatic activities of NTPDase and ADA in serum of hypercholesterolemic rats, in a concentration-dependent manner. These results indicated that ß-caryophyllene treatment could improve the immune response during hypercholesterolemia through purinergic pathway.

Purinergic ecto-enzymes participate in the thromboregulation in acute in mice infection by Trypanosoma cruzi.

Coagulation disorders have been described in Chagas disease with thrombocytopenia as an important event. Several mechanisms may be related to this pathogenesis, such as enzymes of the purinergic system, purine, and receptors involved in the regulation and modulation of physiological events related to hemostasis. Therefore, the aim of this study was to evaluate the activities of E-NTPDase, E-5'nucleotidase, and ecto-adenosine deaminase (E-ADA) in platelets of mice experimentally infected by Trypanosoma cruzi. Twelve female mice were used, divided into two groups (n = 6): uninfected and infected. Mice of infected group were intraperitoneally inoculated with 104 trypomastigotes of T. cruzi (strain Y). On day 12 post-infection (PI), blood samples were collected for quantitation and separation of platelets. A significant reduction in the number of platelets of infected mice (P < 0.05) was observed. The activities of E-NTPDase (ATP and ADP substrates), E-5'nucleotidase, and E-ADA in platelets increased significantly (P < 0.05) in mice infected by T. cruzi compared with uninfected animals. A negative correlation (P < 0.01)was observed between the number of platelets and ATP hydrolysis (r = -0.64), and ADP hydrolysis (r = -0.69) by E-NTPDase. Therefore, there is a response from the purinergic system activating ecto-enzymes in platelets of mice T. cruzi infected, as a compensatory effect of thrombocytopenia.

Diphenyl diselenide modulates nucleotidases, reducing inflammatory responses in the liver of Toxoplasma gondii-infected mice.

The aim of this study was to verify the effect of diphenyl diselenide (PhSe)2 on hepatic nucleotidases and on the concentration of purines in mice infected by Toxoplasma gondii. The animals were divided into four groups: Group A (uninfected), Group B (uninfected and treated with (PhSe)2), Group C (infected), and Group D (infected and treated with (PhSe)2). The inoculation (groups C and D) was performed with 50 cysts of T. gondii (ME-49 strain). Mice from groups B and D were treated with 5 µmol kg-1 of (PhSe)2. Liver tissue from infected mice showed less severe inflammation, elevated ATP/ADO ratio, elevated NTPDase, 5'nucleotidase, and ADA activities compared to the uninfected group (Group A; P < 0.05). However, infected and treated mice showed decreased ATP levels and elevated ADO levels, as well as higher NTPDase and 5'nucleotidase activities and decreased ADA activity in the hepatic tissue compared to the infected group (P < 0.05). Moreover, the (PhSe)2 treatment of infected mice reduced the hepatic inflammation and showed an immunomodulatory effect on ectonucleotidases of hepatic lymphocytes, which it returned to basal levels. Therefore, chronic infection by T. gondii induces hepatic inflammation in mice, and it is possible that purine levels and nucleotidase activities in hepatic tissue are related to the pathogenesis of the infection in this tissue. The treatment with (PhSe)2 was able to reverse the hepatic inflammation in mice chronically infected, possibly due to the modulation of purinergic enzymes that produce an anti-inflammatory profile through the purinergic system in the liver tissue.

Characterization of E-NTPDase (EC 3.6.1.5) activity in hepatic lymphocytes: A different activity profile from peripheral lymphocytes.

The activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) was characterized in hepatic lymphocytes (HL) of rats. For this purpose, a specific method for the isolation of lymphocytes from hepatic tissue was developed. Subsequently, E-NTPDase activity of rat HL was compared with that of rat peripheral lymphocytes. The HL showed high cell count and viability. Also, the characterization test revealed that the optimal E-NTPDase activities were attained at 37°C and pH 8.0 in the presence of Ca2+ . In addition, in the presence of specific E-NTPDase inhibitors (20mM sodium azide and 0.3mM suramin), there were significant inhibitions in nucleotide hydrolysis. However, there was no significant change in adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolysis in the presence of inhibitors of other E-ATPase (0.1mM Ouabain, 0.5mM orthovanadate, and 1mM, 5mM, and 10mM sodium azide). Furthermore, the kinetic behavior of the enzyme in HL showed apparent Km of 134.90 ± 0.03µM and 214.40 ± 0.06µM as well as Vmax of 345.0 ± 28.32 and 242.0 ± 27.55 Æžmol Pi/min/mg of protein for ATP and ADP, respectively. The Chevillard plot revealed that ATP and ADP were hydrolyzed at the same active site of the enzyme. Our results suggest that the degradation of extracellular nucleotides in HL may have been primarily accomplished by E-NTPDase. The higher E-NTPDase activity observed in HL may be attributed to the important physiological functions of ATP and ADP in HL. SIGNIFICANCE OF THE STUDY: Extracellular purine nucleotides are able to interact with specific receptors and trigger a number of important physiological functions in cells. This interaction is controlled by ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), enzyme that present their catalytic site at the extracellular space and degrades nucleotides. This purinergic signaling has important functions in peripheral lymphocytes and may represent an important new therapeutic target for the treatment of immunological diseases. However, there is dearth of information on the involvement of E-NTPDase in liver lymphocytes. The liver is an important organ, which performs both metabolic and toxicological roles in living organism, and hepatic lymphocytes may play crucial action in the regulation of immune responses in the liver tissue. Furthermore, various chronic diseases such as cirrhosis may be treated with novel pharmacotherapy by targeting the modulation of hepatic lymphocytes. Thus, the significance of this study is to evaluate the activity of E-NTPDase in liver lymphocyte and compare its activity with the peripheral lymphocytes.

Toxoplasmosis treatment with diphenyl diselenide in infected mice modulates the activity of purinergic enzymes and reduces inflammation in spleen.

Toxoplasma gondii, an intracellular protozoan, may cause chronic infection in the brain tissue of the host inducing a systemic pro-inflammatory profile. Chronic infections can induce numerous physiological changes, such as alterations in the immune and oxidative profiles. Diphenyl diselenide (PhSe)2, an organoselenium compound, has shown antioxidant and immunomodulatory activities in recent studies. So, the aim of this study was to investigate the activity of purinergic enzymes and reactive oxygen species (ROS) in serum and spleen of mice chronically infected by T. gondii, untreated and treated with (PhSe)2. For this experiment, were divided into four groups: Group A (healthy mice), Group B (healthy mice treated with (PhSe)2), Group C (infected mice) and Group D (infected mice treated with (PhSe)2). Group C and group D were infected via oral route with ME49 Toxoplasma gondii strain. Groups B and D were treated subcutaneously with 5 µmol kg-1 of (PhSe)2. Chronic T. gondii infection induced splenomegaly and physiological changes in the spleen and raised histologic inflammatory markers, ROS levels and the activity of purinergic enzymes activity such as NTPDase, 5´nucleotidase and ADA. In serum, the infection increased 5´nucleotidase and ADA activities. (PhSe)2per se has managed to decrease ROS levels and ADA activity and increase NTPDase and 5´nucleotidase in spleen. In infected mice, treatment with (PhSe)2 reversed splenomegaly, reduced histological inflammatory markers, ROS levels and ADA activity in the spleen. Our results prove that chronic toxoplasmosis can induce splenomegaly, heightens ROS levels and purinergic enzyme activity in mice. These results suggest that (PhSe)2 is a potential therapy for the alterations found in the spleen in chronic T. gondii infection.

Purinergic enzymatic activities in lymphocytes and cardiomyocytes of mice acutely infected by Trypanosoma cruzi modulating the inflammatory responses.

The aim of this study was to evaluate the activity of purinergic enzymes in lymphocytes and cardiac tissue of mice experimentally infected by Trypanosoma cruzi. Twelve female mice were used, divided into two groups (n = 6): uninfected and infected. On day 12 post-infection (PI), the animals were anesthetized and after euthanized, and samples were collected for analyses. Infected mice showed reduction in erythrocyte counts, hematocrit and hemoglobin concentration, as well as reduced number of total leukocytes in consequence of neutrophilia (P < 0.01). The number of monocytes increased in infected mice (P < 0.001), however the number of lymphocytes and eosinophils did not differ between groups (P > 0.05). The E-NTPDase (ATP and ADP substrate) and E-ADA activities in lymphocytes increased significantly in mice infected by T. cruzi (P < 0.01). In the heart, multiple pseudocysts containing amastigotes within cardiomyocytes were observed, as well as focally extensive severe necrosis associated with diffuse moderate to severe inflammatory infiltrate of lymphocytes. Although, the NTPDase activity (ATP and ADP substrate) in the cardiac homogenate did not differ between groups, a reduction on 5'-nucleotidase activity (P < 0.001) and an increase in the ADA activity in infected animals (P < 0.05) were observed. Thus, animals infected by T. cruzi experienced the disease, i.e., showed anemia, leucopenia, and heart lesions. Associated with this, purinergic enzymes showed altered activities, which might be related to the modulation of the inflammatory response.

Ectonucleotidases and adenosine deaminase activity in laying hens naturally infected by Salmonella Gallinarum and their effects on the pathogenesis of the disease.

Salmonella Gallinarum is the etiologic agent of fowl typhoid that affects chickens and turkeys causing egg production drops, infertility, lower hatchability, high mortality, and as a consequence severe economic losses to the poultry industry. The alterations in NTPDase and adenosine deaminase (ADA) activities have been demonstrated in several inflammatory conditions; however, there are no data in the literature associated with this infection. Thus, the aim of this study was to evaluate the activities of NTPDase, 5'nucleotidase, and ADA in serum and hepatic tissue of laying hens naturally infected by Salmonella Gallinarum. Liver and serum samples were collected of 27 laying hens (20 S. Gallinarum infected and 7 uninfected). NTPDase and 5'-nucleotidase activities in serum were increased (P < 0.001) in infected animals to hydrolysis of substrate ATP, ADP and AMP. In addition, it was observed decreased (P < 0.001) in ADA activity in serum of laying hens naturally infected by S. Gallinarum; as well as increased (P < 0.001) ADA activity in liver tissue of infected laying hens. Histopathological analyses revealed that S. Gallinarum caused fibrinoid necrosis in liver and spleen associated with infiltrates of heterophils, macrophages, lymphocytes, and plasma cells. Considering that NTPDase and ADA are involved in the cell-mediated immunity, this study suggests that activities of these enzymes could be important biomarkers to determine the severity of inflammatory and immune responses in salmonellosis, contributing to clarify the pathogenesis of the disease.

Influence of experimental Anaplasma marginale infection and splenectomy on NTPDase and 5'nucleotidase activities in platelets of cattle.

The objective of this paper was to evaluate NTPDase and 5'-nucleotidase activities in platelets of bovine with and without spleen and infected by Anaplasma marginale. Our results demonstrate that infection along with splenectomy is able of inducing a profile of cellular protection, which showed an increase in the degradation of the nucleotides ATP and ADP by NTPDase, in addition to AMP by 5'nucleotidase to form the nucleoside adenosine in platelets, i.e., the enzymatic activities of platelets were increased in splenectomized animals when compared to non-splenectomized group. It notes that adenosine is a molecule with anti-inflammatory function. But this profile is related to a deficiency in immune signaling triggered by nucleotide ATP, which may be related to the increase in bacteremia and disability in combating the parasite in splenectomized host.

Cattle experimentally infected by Anaplasma marginale: Influence of splenectomy on disease pathogenesis, oxidative profile, and antioxidant status.

Bovine anaplasmosis is caused by the obligate intraerythrocytic bacteria Anaplasma marginale. These bacteria are transmitted by tick species such as Rhipicephalus (Boophilus) microplus, blood-sucking insects, and fomites (needles, clippers, and other blood contaminated equipment). During the acute phase of infection, animals may develop fever, anemia, jaundice, and hepatosplenomegaly. The aims of this study are to quantify the bacteremia by quantitative PCR in eight naïve calves experimentally infected by A. marginale [splenectomized (n = 4), and intact/non-splenectomized (n = 4)], and to correlate these findings with markers of oxidative stress on days 0, 8, 15, 21 and 23 post-infection. Complete blood counts (CBC) were performed in both groups. Lipid peroxidation was estimated by quantifying thiobarbituric acid reactive substances (TBARS); and non-enzymatic antioxidants were assessed by erythrocyte content of non-protein thiols (NPSH). There were no significant differences in complete blood counts (CBC) between the two groups. However, both groups had a slight decrease on packet cell volume (PCV), erythrocytes and hemoglobin concentration, as well as an increase in total leukocyte counts due to elevated lymphocytes when comparing pre and post-infection with A. marginale. Progressive increase on TBARS levels and concomitant decrease on NPSH content were observed in all animals, without significant differences between splenectomized and intact animals. A positive correlation between bacteremia and TBARS, and a negative correlation between bacteremia and NPSH were observed in both groups with higher correlation for NPSH in splenectomized animals. A negative correlation between TBARS and NPSH levels was observed in both groups indicating lipid peroxidation without a non-enzymatic antioxidant response. The results of experimental infection by A. marginale in cattle showed that bacteremia has an impact on lipid peroxidation regardless of the splenectomy.