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Rheumatoid arthritis (RA) is a classic inflammatory autoimmune disease. Local joint destruction and extra-articular manifestations of RA deeply compromise the life quality of the affected patients. RA immunopathogenesis depends on continuous immunogenic activation in which the purinergic system participates. The purinergic system comprises the signaling and metabolism of purines such as adenosine triphosphate (ATP) and adenosine. ATP signaling is involved in the activation and maintenance of the inflammatory state of RA through the activation of P2X7 and the production of cytokines, which orchestrate the pathogenesis of RA. The breakdown of ATP through the CD39/CD73 axis produces adenosine, which mostly inhibits the inflammatory process through activation of specific P1 receptors. Adenosine is hydrolyzed by adenosine deaminase (ADA) that interacts with other molecules playing additional roles in this disease. This review explores the release, metabolism, and the effects of binding of ATP and adenosine to their respective receptors in the context of RA, as well as their potential use as biomarkers and therapeutic targets.

Systemic lupus erythematosus (SLE) is an autoimmune and inflammatory disease with periods of exacerbation and remission. SLE is characterized by the irreversible breakdown of immunological self-tolerance, where there is deregulation of multiple aspects of the immune system. SLE immune dysfunction is characterized by activation of autoreactive T lymphocytes, and hyperactivity of B lymphocytes with consequent production of several autoantibodies. ATP is a purinergic mediator released into the extracellular space in response to cell and tissue damage which operates as a danger signal to modulate immune and inflammatory responses. ATP binds to P2 receptors and its levels are regulated by NTPDase (CD39). SLE patients exhibit increased levels of ATP which binds to P2X receptors resulting in activation of the inflammasome and consequent release of IL-1β and IL-18, cytokines associated with disease pathogenesis. CD39 is upregulated in SLE representing an important immunoregulatory mechanism by controlling inflammation and favoring the production of adenosine. The aim of this review is to clarify the effects of ATP on the modulation of the inflammatory process and immune responses via P2 receptors as well as the role of NTPDase in the immunopathogenesis of SLE.

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) 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.

The aim of this study was to evaluate the enzymatic activity of the purinergic system in sera samples from alloxan-induced diabetic mice treated with tucuma oil (Astrocaryum vulgare). For this, the mice were divided into four groups (n = 6): control/water (the group CW); control/tucuma oil (the group CT); diabetic/water (the group DW), and diabetic/tucuma oil (the group DT) treated for 14 days with 5.0 mL kg−1 via oral gavage. On day 14 post-treatment, mice were submitted to euthanasia and blood samples were collected by cardiac puncture. Tucuma oil treatment significantly decreased (p

The aim of this study was to evaluate the influence of dictyocaulosis (mild or severe) on enzymes of NTPDase, 5'-nucleotidase, and adenosine deaminase (ADA) of dairy cows naturally infected by Dictyocaulus viviparus. Blood and faeces were collected from 22 dairy cows of the same farm to evaluate NTPDase (ATP and ADP substrate), 5'-nucleotidase, and ADA activities on days 0 (pre-treatment) and 10 (post-treatment). Seric activities of NTPDase (ATP substrate), 5'-nucleotidase, and ADA were lower (P0.05) in D. viviparus infected animals compared to uninfected cows. The number of D. viviparus larvae per gram of faeces varied among the animals, and they showed different degrees of severity according to respiratory clinical signs of the disease (cough and nasal discharge). Later, these cows were divided into two groups: those with mild (n=10) and severe (n=12) disease. Cows with severe disease showed higher NTPDase activity (ATP substrate) than those with mild disease (P≤0.05). The opposite occurred with NTPDase (ADP substrate), 5'-nucleotidase, and ADA in cows with severe disease, that is, the enzymatic activity of these seric enzymes significantly decreased (P≤0.05) compared to animals with mild disease. Infected animals showed reduced NTPDase activity (ATP and ADP substrate) after treatment. No enzymatic changes were observed for 5'-nucleotidase, and ADA pre- and post-treatment (P0.05). Based on these results, we conclude that dictyocaulosis alters NTPDase, 5'-nucleotidase, and ADA activities of cow naturally infected by the parasite, in consequence the enzymes act as inflammatory markers.

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.

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

The aim of this study was to evaluate seric NTPDase and 5′nucleotidase activities of cattle naturally infected by Eurytrema coelomanticum, as well as to correlate them to histopathological lesions in the pancreas and the degree of parasitism. Blood samples and pancreas of 51 bovines were collected on a slaughterhouse in Southern Brazil: 33 from cattle naturally infected by E. coelomanticum (the Group A), and 18 from uninfected animals (the Group B). Infected animals showed an average of 532 parasites per pancreas. In the pancreatic histology, ducts displayed hyperplasia, stenosis, proliferation of fibrous tissue, and interstitial inflammatory infiltration of lymphocytes. The serum from infected animals showed an increase in NTPDase activity when ATP was used as substrate (P < 0.001). For the ADP substrate, there was no difference between groups regarding NTPDase activity (P = 0.37), as well as 5′-nucleotidase activity (P = 0.27). Correlating NTPDase activity (ATP substrate) with the degree of histopathological lesions (rho = 0.66, P < 0.001) and the parasitic load on the pancreas (rho = 0.65, P < 0.001), a positive correlation was observed. Similar results were found between the degree of histopathological lesions and NTPDase activity (ADP substrate; rho = 0.29, P = 0.03), and 5′nucleotidase activity (rho = 0.35, P = 0.01). Based on the results of NTPDase and 5′nucleotidase enzymes in cattle naturally infected by E. coleomanticum, it is possible to suggest that these enzymes are involved in the modulation of inflammation, and they can act as markers of inflammatory response.

The neurotoxic effects and activity of Na(+), K(+)-ATPase and NTPDase in Wistar rats after treatment with α-terpinene (daily oral administration of 0.5, 0.75 and 1.0mLkg(-1) for 10days) were examined. Results of the inhibitory avoidance task showed a memory deficit (p

The effects of chlorogenic acid (one of the major phenolic acid found in human diets) were investigated on the adenine nucleotides hydrolyzing enzymes; ecto-nucleotide pyrophosphatase/phophodiesterase (E-NPP), ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'- nucleotidase and ecto-adenosine deaminase (E-ADA) activities and expression in platelets of rats experimentally demyelinated with ethidium bromide.Rats were divided into four groups of eight animals each. Group I rats were control rats; injected with saline (CT), group II rats were injected with saline and treated with chlorogenic acid (AC), group III rats were injected with 0.1% ethidium bromide (EB) and group IV rats were injected with 0.1% EB and treated with chlorogenic acid (EB+AC). The activities of the enzymes were analyzed using colorimetric methods, and the gene expression of NTPDase 1, 2 and 3 were analyzed using the polymerase chain reaction (PCR).The results revealed that there was a significant (P0.01) reduction in E-NPP activity in EB group (1.63±0.10nmol p-nitrophenol released/min/mg protein) when compared to CT group (2.33±0.14nmol p-nitrophenol released/min/mg protein). However, treatment with chlorogenic acid significantly (P0.05) increased E-NPP activity in EB group. Furthermore, no significant (P0.05) change was observed in the E-NPP activity of EB+AC group (2.19±0.08nmol p-nitrophenol released/min/mg protein) when compared to CT group (2.33±0.14nmol p-nitrophenol released/min/mg protein). In addition, there was a significant (P0.05) increase in AMP hydrolysis in EB rat group when compared to CT group. No significant (P0.05) difference was observed in AMP hydrolysis between AC, AC+EB and CT groups. Conversely, there were no significant (P0.05) differences in ATP and ADP hydrolyses between all the groups (AC, EB, AC+EB and CT groups). Likewise, there were no significant (P0.05) changes in E-ADA activity and percentage platelet aggregation among all groups studied. Similarly, no significant (P0.05) change was observed in the expression of E-NTPDase 1, 2 and 3 in all the groups tested.Our study revealed that chlorogenic acid may modulate the hydrolysis of adenine nucleotides in platelets of rats demyelinated and treated with chlorogenic acid via alteration of E-NPP and ecto-5'-nucleotidase activities.

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.

Sickle cell anemia (SCA) is a hemoglobinopathy characterized by hemolysis and vaso-occlusions caused by rigidly distorted red blood cells. Sickle cell crisis is associated with extracellular release of nucleotides and platelets, which are critical mediators of hemostasis participating actively in purinergic thromboregulatory enzymes system.This study aimed to investigate the activities of purinergic system ecto-enzymes present on the platelet surface as well as CD39 and CD73 expressions on platelets of SCA treated patients. Fifteen SCA treated patients and 30 health subjects (control group) were selected. Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5'-nucleotidase (E-5'-NT) and ecto-adenosine deaminase (E-ADA) activities were measured in platelets isolated from these individuals. Results demonstrated an increase of 41 % in the E-NTPDase for ATP hydrolysis, 52% for ADP hydrolysis and 60 % in the E-ADA activity in SCA patients (P0.05); however, a two folds decrease in the CD39 expression in platelets was observed in the same group (P0.01). The increased E-NTPDase activity could be a compensatory mechanism associated with the low expression of CD39 in platelets. Besides, alteration of these enzymes activities suggests that the purinergic system could be involved in the thromboregulatory process in SCA patients.

The aim of this study was to evaluate the effects of treatment with free and nanoencapsulated essential oil of Achyrocline satureioides on trypanosomosis and its oxidative/antioxidants variables in liver and kidney of rats infected experimentally with Trypanosoma evansi. For that, 48 rats were divided into six groups (A-F), eight animals each group. Groups A, C and D were composed of uninfected animals, while animals in groups B, E and F were inoculated intraperitoneally with T. evansi. Groups A and B were used as controls, negative and positive, respectively. Groups C and E receive oil (orally), as well as the animals in groups D and F were treated with nanoencapsulated essential oil. The treatment was not able to eliminate the parasites, but it remained the levels of parasitemia low. The carbonyl levels in liver and kidney did not differ between groups. Infected animals (group B) showed an increase in the TBARS levels and a decrease in the CAT activity and NPSH levels in liver and kidney, compared with the same parameters in the control (group A). Treatment with A. satureioides (groups C and D) did not influence the TBARS levels and CAT activity in the liver, but it increased the CAT activity in kidneys of the animals of group C. NPSH levels decreased in liver in the groups treated with nanoencapsulated essential oil (groups D and F). An interesting result observed was that the animals infected and then treated with essential oil of A. satureioides (groups E and F) did not differ from animals of group A for TBARS, CAT and NPSH, unlike what happened with the animals of group B. Therefore, the treatment with essential oil did not eliminate the parasites from the bloodstream, but it reduced the number of trypanosomes, mainly by its nanoencapsulated form. The same occurred with the lipid peroxidation in the liver. However, the treatments reduced the oxidative damage, and it led to the activation of the antioxidant enzymes. We believe that the association of this natural product with a trypanocidal drug may enhance its curative effect.

Cryptococcus neoformans, the etiological agent of cryptococcosis, is an opportunistic fungal pathogen of immunocompromised individuals. The aim of this study was to evaluate the activities of E-NTPDase and E-ADA in rats experimentally infected by C. neoformans var. grubii. Adult rats (35) were divided in two groups: 18 for the control group (uninfected) (A), and 17 for the infected group (B). Each group was separated into three sub-groups (A1, A2, A3-B1, B2, B3), and samples were collected on 10, 20, and 30 days post-infection (PI). Leukocyte counts, IFN-γ, TNF-α, IgM, IgG levels, and E-NTPDase and E-ADA activities were analyzed. It was possible to observe that IgG and IgM seric levels of infected rats were significantly elevated (P

This study investigated the δ-aminolevulinate dehydratase (δ-ALA-D) activity in whole blood as well as the parameters of oxidative stress, such as reactive species (RS) levels in serum, thiobarbituric acid reactive substances (TBARS) levels, the superoxide dismutase (SOD) and catalase (CAT) activities, as well as total thiols (T-SH) and non-protein thiols (NPSH) levels in platelets. Moreover, the content of vitamin C and E in plasma and serum, respectively, in lung cancer patients was also investigated. We collected blood samples from patients (n=28) previously treated for lung cancer with chemotherapy. Patients were classified as stage IIIb and IV according to the Union for International Cancer Control (UICC). Results showed a decrease of 37% in δ-ALA-D activity in patients with lung cancer when compared to the control group. RS and TBARS levels were 8% and 99% higher in the patient group, respectively. The activity of SOD and CAT as well as the vitamin C content were 41%, 35% and 127% lower in patients when compared with controls, respectively. However, T-SH and vitamin E levels were 27% and 44% higher in lung cancer patients, respectively. Results show that the overproduction of reactive species in patients with lung cancer may be interfering with the activity of δ-ALA-D. Likewise, the decrease in the activity of this enzyme may be contributing for the oxidative stress.

This study aimed to investigate the butyrylcholinesterase (BChE) activity in mice experimentally infected with Toxoplasma gondii during the acute phase. Twenty mice were divided in two groups with 10 animals each: group A was composed of uninfected mice while group B was formed by rodents infected with T. gondii. Five days after infection, blood was collected and serum separated, and fragments of liver and brain were obtained. In serum and liver homogenate was noted a significant reduction (P0.05) in BChE activity in infected mice when compared with uninfected ones. In serum was observed an increase in the activity of alanine aminotransferase and urea, associated with reduction in alkaline phosphatase activity and in the levels of total protein and albumin. Histologically, there were foci of necrosis and parasites in the forms of tachyzoites and cysts, with bradyzoites in liver samples of infected animals. Based on these results, we conclude that toxoplasmosis reduces BChE activity in mice, and this alteration is probably related to the liver damage caused by the parasitism. Therefore, this enzymatic alteration can directly contribute to the pathogenesis of the disease.

Trypanosoma cruzi infection triggers a chronic inflammatory process in human host and purinergic system ecto-enzymes play an important role in modulating the inflammatory and immune responses. In this study, it was investigated ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5; CD39) and ecto-adenosine deaminase (E-ADA; EC 3.5.4.4) activities in lymphocytes from patients with indeterminate form of Chagas' disease (IFCD). Twenty-five IFCD patients and 25 healthy subjects (control group) were selected. The peripheral lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Adenine nucleotides and adenosine levels were determined in serum by HPLC and the E-NTPDase1 expression in lymphocytes by Western blot analysis. E-NTPDase (ATP and ADP as substrates) and E-ADA (adenosine as substrate) activities were decreased in lymphocytes from IFCD patients (P0.05 and P0.01, respectively), while the E-NTPDase1 expression presented no changes in these patients. Serum ATP levels showed to be decreased (P0.05) and both AMP (P0.01) and adenosine (P0.001) levels were increased in the IFCD group. The enzymatic alterations observed are in agreement with the immune response against T. cruzi infection in IFCD patients, since the decreased extracellular ATP and the increased adenosine levels trigger a Th2 anti-inflammatory response, which it is associated to adaptation of host to parasite, preventing clinical progress of disease.

Cigarette smoke, a widely spread habit, is associated with a decline in cognitive function and studies have demonstrated that curcumin (Cur), an Indian spice, possesses a strong neuroprotective potential. Considering the relevance of investigating dietary compounds this study aimed to investigate the effect of Cur on memory and acetylcholinesterase (AChE) activity in brain structures and blood of cigarette smoke-exposed rats. Male Wistar rats were treated with curcumin and cigarette smoke, once a day, 5 days each week, for 30 days. The experimental procedures were divided in two sets of experiments. In the first, the animals were divided into 4 groups: Vehicle (corn oil), Cur 12.5 mg/kg, Cur 25 mg/kg and Cur 50 mg/kg. In the second, the animals were divided into 5 groups: Vehicle (corn oil), Smoke, Smoke plus Cur 12.5 mg/kg, Smoke plus Cur 25 mg/kg and Smoke plus Cur 50 mg/kg. Treatment with Cur significantly prevented the decreased latency and cholinergic alterations in cigarette smoke-exposed rats. These AChE alterations could suggest a role in the memory impairment promoted by cigarette smoke-exposure and point toward the potential of Cur to modulate cholinergic neurotransmission and, consequently, improve cognition deficits induced by smoke. This study suggests that the dietary compound Cur may be involved in cholinergic system modulation and as a consequence exert an effect on learning and memory.

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 (P0.05). 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.

Background The nucleotides and nucleosides of adenine are signaling molecules related to thromboregulation and modulation of immune responses in patients with malignancies. Thus, this study aims to determine NTPDase, 5′-nucleotidase, ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) activities in the platelets of patients with lung cancer. Methods We collected blood samples from patients ( n = 33) previously treated for lung cancer with chemotherapy. Patients were classified as stage IIIb and IV according to the Union for International Cancer Control (UICC). Results Patients showed a significant decrease in the hydrolysis of adenosine diphosphate (ADP) and adenosine, whereas the adenosine monophosphate (AMP) hydrolysis and platelet aggregation were significantly increased in this group. Adenosine triphosphate (ATP) hydrolysis did not show significant results between the group of patients and the control group. Conclusions We may suggest that ectonucleotidases as well as ADA are enzymes involved in thromboembolic events but especially here we may see that they are also directly involved in the generation of adenosine formation in the cancer patient circulation.

Nucleotide and nucleoside-degrading enzymes, such as nucleoside triphosphate diphosphohydrose (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA) are present in the surface membranes of platelets, involved in clotting disturbances of Trypanosoma evansi-infected animals. Thus, this study was aimed at evaluating the activities of these enzymes in platelets of rats experimentally infected with T. evansi. Animals were divided into four groups, according to the level of parasitemia. Blood samples were collected on days 3 (group A: at the beginning of parasitemia), 5 (group B: high parasitemia) and 15 (group C: chronic infection), post-infection. Group D (control group) was composed of non-infected animals for platelet count, separation and enzymatic assays. Animals from groups A and B showed marked thrombocytopenia, but platelet count was not affected in chronically infected rats. NTPDase, 5'-nucleotidase and ADA activities decreased (p0.05) in platelets from rats of groups A and B, when compared to the control group. In group C, only NTPDase and 5'-nucleoside activities decreased (p0.001). The correlations between platelet count and nucleotide/nucleoside hydrolysis were positive and statistically significant (p0.05) in groups A and B. Platelet aggregation was decreased in all infected groups, in comparison to the control group (p0.05). It is concluded that the alterations observed in the activities of NTPDase, 5'-nucleotidase and ADA in platelets of T. evansi-infected animals might be related to thrombocytopenia, that by reducing the number of platelets, there was less release of ATP and ADP. Another possibility being suggested is that changes have occurred in the membrane of these cells, decreasing the expression of these enzymes in the cell membrane.

The aim of the present study was to evaluate the activity of the enzymes NTPDase (EC 3.6.1.5) and acetylcholinesterase (AChE; EC 3.1.1.7) in lung lymphocytes (LL) and peripheral lymphocytes (PL) from cigarette smoke-exposed rats treated with curcumin (Cur). The animals were treated with cigarette smoke and curcumin, once a day, 5 days each week. The experimental procedures were divided into two sets of experiments. In the first set, the animals were distributed into four groups: vehicle, Cur 12.5, Cur 25 and Cur 50 mg/kg. In the second set, the animals were divided into five groups: vehicle, smoke, smoke and Cur 12.5 mg/kg, smoke and Cur 25 mg/kg, smoke and Cur 50 mg/kg. After 30 days, the lung was removed and the peripheral blood collected for separation of lymphocytes. The treatment with curcumin prevented alterations observed in the cigarette smoke-exposed animals such as the decrease of ATP and ADP hydrolysis in PL and LL, and the increase of AChE activity in PL. We suggest that the treatment with curcumin was protective, since the high concentrations of ATP are positively related with inflammation and tissue damage, and the histological injury observed in cigarette smoke-exposed rats was not observed in the groups treated with curcumin.

Methods for the isolation of peripheral blood mononuclear cells (PBMCs) and human lung mononuclear cells (LMCs) have been proposed previously. This study describes a method that allows the separation of lymphocyte-rich LMCs from rats. Trypan blue was applied to determine cell viability. White blood cell and differential cell counts were also performed. Relationships between nucleoside triphosphate diphosphohydrolase (NTPDase, EC 3.6.1.5) activities expressed in milligrams of protein, millions of cells, and millions of viable cells were examined as linear correlations. The lung tissue yielded 82.46% lymphocytes, 8.6% macrophages, 2.20% monocytes, and 1.27% polymorphonuclear cells (PMNs). In LMCs, a very strong correlation was observed as follows: between NTPDase activity, as determined using ATP or ADP as a substrate, expressed in milligrams of protein and that expressed in millions of cells (r ≥ 0.91), between that expressed in milligrams of protein and that expressed in millions of viable cells (r ≥ 0.91), and between that expressed in millions of cells and that expressed in millions of viable cells (r ≥ 0.98). Based on our results, we affirm that NTPDase activity could be expressed in millions of viable cells, millions of cells, or milligrams of protein.

Infection with the human immunodeficiency virus (HIV) results in alterations in immune cells such as an increase or decrease of cytokine secretion and immunodeficiency. HIV causes a state of chronic cellular activation that can induce apoptosis in lymphocyte T-helpers, making the patient susceptive to opportunistic infections. The biochemical mechanisms involved in this immune response to HIV have been researched. Here, we have shown for the first time that ATP and ADP hydrolysis are essential for the immune response to HIV. Our results clearly indicate an increase of NTPDase-1 (EC 3.6.1.5) activity in lymphocytes of HIV-positive patients, confirmed by an enhanced CD39 expression on its surface. These results suggest that NTPDase-1 may be important to keep an adequate balance between the generation and consumption of ATP and to preserve cellular integrity and immune response to the HIV infection.

Human lymphocytes contain NTPDase (NTPDase-1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5), a cation-dependent enzyme that hydrolyzes ATP and ADP and also other di- and triphosphate nucleosides, acting at an optimum pH of 8.0. A significant inhibition of ATP and ADP hydrolysis (P0.05) was observed in the presence of 20 mM sodium azide. NTPDase inhibitors, 20 mM sodium fluoride, 0.2 mM trifluoperazine and 0.3 mM suramin, significantly decreased ATP and ADP hydrolysis (P0.05) and ADP hydrolysis was only inhibited by 0.5 mM orthovanadate (P0.05). ATP and ADP hydrolysis was not inhibited in the presence of 0.01 mM Ap5A (P1,P5-di(adenosine-5')pentaphosphate), 0.1 mM ouabain, 1 mM levamisole, 2 microg/mL oligomycin, 0.1 mM N-ethylmaleimide (NEM), or 5 mM sodium azide. With respect to kinetic behavior, apparent K(m) values of 77.6+/-10.2 and 106.8+/-21.0 microM, and V(max) values of 68.9+/-8.1 and 99.4+/-8.5 (mean+/-S.E., n=3) nmol Pi/min/mg protein were obtained for ATP and ADP, respectively. A Chevilard plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. The presence of CD39 was determined by flow cytometry, showing a low density of 2.72+/-0.24% (mean+/-S.E.; n=30) in human peripheral lymphocytes. The study of NTPDase activity in human lymphocytes may be important to determine the immune response status against infectious agents related to ATP and ADP hydrolysis.

NTPDase (EC 3.6.1.5) is an enzyme that hydrolyzes extracellular nucleoside tri- and/or diphoshates forming AMP that can serve as a substrate for an ecto-5′-nucleotidase (EC 3.1.3.5) with liberation of adenosine, a modulator of vascular tone and inhibitor of platelet aggregation. These enzymes also occur in lymphocytes playing an important role in immune function. In this study, it was investigated if anti-HIV therapy could affect NTPDase activity in human lymphocytes. Samples of lymphocytes were incubated with different concentrations of anti-HIV drugs and NTPDase activity was determined by colorimetric assay with quantification of inorganic phosphate released. There is not significant difference of NTPDase activity among samples with therapeutic doses of anti-HIV drugs tested when compared with controls. NTPDase activity in peripheral human lymphocytes is not altered by anti-HIV therapy.