Diagnostic utility of the basophil activation test in natto-induced hypersensitivity.

BACKGROUND: Natto (fermented soybeans)-induced hypersensitivity is characterized by delayed symptom onset that hampers diagnosis. We aimed to clarify the clinical utility of the basophil activation test (BAT) in the diagnosis of natto-induced hypersensitivity. METHODS: Five patients with a history of anaphylaxis and chronic urticaria suspected of natto-induced hypersensitivity and seven with chronic spontaneous urticaria clinically unrelated to natto were enrolled in the patient and control groups, respectively. The BAT was performed with two incubation times, 15 min and 1 h, in combination with various concentrations of natto-mucilage extract. RESULTS: In controls, CD203c levels in basophils remained low in the 15-min incubation but were significantly increased in the 1-h incubation. In the patient group, in the 15-min condition, basophil CD203c was significantly upregulated by natto mucilage but not by soybean vs controls (P = 0.001). Low concentrations of natto mucilage were sufficient to upregulate basophil CD203c in the anaphylaxis cases, but high concentrations were required to induce the same effect in the urticaria cases. Finally, the dose-dependent pattern of the BAT results differed significantly between the anaphylaxis and urticaria cases (P = 0.006). Thus, a strong background reaction was observed in the BAT with 1 h incubation; 15 min of incubation was sufficient to identify patients with natto-induced hypersensitivity and may distinguish the clinical phenotype of natto-induced hypersensitivity, i.e., anaphylaxis or urticaria. CONCLUSIONS: The BAT with a 15-min incubation period is useful in diagnosing natto-induced hypersensitivity.

Serum CD203c+ Extracellular Vesicle Serves as a Novel Diagnostic and Prognostic Biomarker for Succinylated Gelatin Induced Perioperative Hypersensitive Reaction.

Background: Perioperative hypersensitivity reaction (HR) is an IgE-FcϵRI-mediated hypersensitivity reaction with degranulation and activation of mast cells and basophils. Several studies have focused on assessing the degranulation and activation of mast cells and basophils to diagnose and predict the prognosis of drug induced HR. However, it is challenging to isolate sufficiently pure mast cells and basophils from human sources to investigate. Effective biomarkers to assess mast cells and basophils activation in vivo could potentially have high diagnostic and prognostic values. In the present study, we investigated EVs pelleted from serum in patients with succinylated gelatin induced HR. Methods: Extracellular vesicles (EVs) were isolated using a total exosome isolation kit and ultracentrifugation, characterized by Western blot, transmission electron microscopy, and nanoparticle tracking analysis. Basophils were isolated from fresh peripheral blood by negative selection using Basophil Isolation Kit II. Human mast cell line was stimulated with IL4. The expression levels of proteins related to the hypersensitive response were evaluated by Western blotting and flow Cytometer. Histamine and tryptase levels were tested using a commercial ELISA kit, and gene expression of inflammatory mediators was evaluated by qRT-PCR. The receiver operating characteristic (ROC) curve was used to evaluate the specificity and sensitivity of biomarker in predicting HR. Results: The concentration of EVs and protein expression level of CD63, FcϵRI, CD203c and tryptase were significantly (p< 0.05) increased in HR samples. The expression level of mast cell/basophil specific CD203c were significantly increased in EVs derived from serum and basophils of HR patients, and the CD203c+-EVs production in mast cells is dramatically increased in the presence of IL4, which positively correlated with histamine, tryptase and inflammatory mediators. Moreover, the ROC curve of EVs concentration and CD203c expression indicated that CD203c+-EVs had a strong diagnostic ability for HR. Conclusion: Serum CD203c+-EVs serves as a novel diagnostic and prognostic biomarker for HR.

Increased DNA-incorporated thiopurine metabolite as a possible mechanism for leukocytopenia through cell apoptosis in inflammatory bowel disease patients with NUDT15 mutation.

BACKGROUND AND AIMS: Polymorphisms in the nucleotide diphosphate-linked moiety X-type motif 15 (NUDT15) gene are associated with thiopurine-induced leukopenia in patients with inflammatory bowel disease (IBD). NUDT15-associated subcellular thiopurine metabolism has not been investigated in primary lymphocytes. We hypothesized that NUDT15 mutation increases DNA-incorporated deoxythioguanosine (dTG) and induces apoptosis in lymphocytes. METHODS: DNA-incorporated dTG in peripheral blood mononuclear cells (PBMCs) and 6-thioguanine nucleotides (6-TGN) in red blood cells were measured in patients with IBD undergoing thiopurine treatment. The association of a single nucleotide polymorphism for NUDT15 (rs116855232) with dTGPBMC was examined. The pro-apoptotic effect of DNA-incorporated dTG was examined ex vivo in association with NUDT15 genotypes by co-culturing patient-derived peripheral CD4+ T lymphocytes with 6-thioguanine (6-TG). RESULTS: dTGPBMC was significantly higher in NUDT15 variants than in non-variants. dTGPBMC, but not 6-TGNRBC, negatively correlated with peripheral lymphocyte counts (r = - 0.31 and - 0.12, p = 0.012 and 0.173, respectively). DNA-incorporated dTG significantly accumulated to a greater extent in lymphocytes from NUDT15 variants when co-cultured with 6-TG ex vivo than in those from non-variants and was associated with decreased proliferation and increased apoptosis. CONCLUSION: Increased DNA-incorporated dTG may be responsible for thiopurine-induced leukocytopenia through cell apoptosis in IBD patients with NUDT15 mutation.

Occupational exposure to metal-rich particulate matter modifies the expression of repair genes in foundry workers.

Foundry workers are exposed to numerous occupational health hazards, which may result in increased risk of cancer, respiratory disease, and other diseases. Oxidative stress is known to be involved in the pathogenesis of such diseases. The present study aimed to investigate the association between multiple occupational exposures in foundry workers and expression of deoxyribonucleic acid (DNA) repair genes as a biomarker of oxidative DNA damage. The study sample comprised 17 foundry workers and 27 matched control subjects. Expression of 8-oxoguanine DNA glycosylase-1 (OGG1), inosine triphosphate pyrophosphate (ITPA), and MutT homolog 1 (MTH1) in peripheral blood was examined using the real-time polymerase chain reaction method. Air sampling to determine exposure to metal-rich particulate matter and measurement of extremely low-frequency electromagnetic fields (ELF-EMFs) were conducted according to the National Institute for Occupational Safety and Health standard methods. Personal air sampling revealed that occupational exposure to particulate matter exceeded the threshold limit values (TLVs) in 76% of the workstations, whereas ELF-EMF exposure appeared to be lower than the TLV. ITPA was significantly upregulated in foundry workers compared with control subjects, whereas no significant difference was observed for OGG1 and MTH1. Moreover, ITPA was strongly and positively correlated with the concentration of metal-rich particulate matter in foundry workers. No significant correlation was found between ELF-EMF exposure and expression of DNA repair genes. DNA repair gene expression may be a sensitive biomarker for occupational exposures, which suggests an involvement of oxidative stress in metal-induced toxicity. Further studies are needed to determine the role of DNA repair gene expression in response to occupational/environmental hazards.

Red Blood cell IMPDH activity in adults and children with or without azathioprine: Relationship between thiopurine metabolites, ITPA and TPMT activities.

Inosine monophosphate dehydrogenase (IMPDH) is considered as the limiting enzyme of thiopurine metabolism for the formation of 6-thioguanine nucleotides (6-TGN). No data are available on the influence of RBC IMPDH activity on the metabolism of thiopurine drugs in individuals with IBD. The aims of this study were as follows: (a) to carry out a phenotypic study of RBC IMPDH activity in adults and children treated or not with azathioprine (AZA) for autoimmune diseases, and (b) to investigate the relationship between the activities of IMPDH, thiopurine metabolites, inosine triphosphatase (ITPA) and thiopurine methyltransferase (TPMT). IMPDH activity was determined in 97 adults and 67 children treated or not with AZA. 6-Thioguanine nucleotides (6-TGN), 6-methylmercaptopurine nucleotide (6-MeMPN) levels, and ITPA as well as TPMT activities were measured in RBCs by HPLC. Using the Gaussian mixture model, distribution of IMPDH activity was evaluated. Influence of age, sex and AZA treatment on IMPDH activity was also assessed. A bimodal distribution in IMPDH activity was found with 87% of patients exhibiting normal activity and 13% of patients with high activity. No influence of age, sex and AZA therapy was found. There is no relationship between TPMT, ITPA and IMPDH activities. A negative correlation between IMPDH activity and 6-MeMPN was shown in adults and children (rs = -0.335 P = 0.014 and rs = -0.383 P = 0.012, respectively). Our results suggest that AZA-treated patients exhibiting lower IMPDH activity could have higher Me-6MPN levels with higher risk of hepatotoxicity. We demonstrated that RBC matrix could be an interesting alternative to lymphocyte matrix to monitor thiopurine metabolites and enzyme activity.

Copper nanoclusters capped with tannic acid as a fluorescent probe for real-time determination of the activity of pyrophosphatase.

The authors show that copper nanoclusters capped with tannic acid (TA-CuNCs) are viable fluorescent probes for the determination of the activity of pyrophosphatase (PPase). The fluorescence of the TA-CuNCs is quenched by Fe(III) but restored on subsequant addition of pyrophosphate (PPi). If, however, PPi is split by PPase into two phosphate ions, the fluorescence of the TA-CuNCs is quenched again. Under optimized conditions, fluorescence intensity linearly correlates with the activity of PPase in the range from 0.50 to 18.0 U L-1 with a detection limit of 0.19 U L-1. The method was employed to the determination of PPase in spiked human serum samples and gave satisfactory results. Graphical abstract Schematic illustration of fluorometric assay for PPase activity. It is based on the finding that pyrophosphate (PPi) can restore the Fe3+-quenched TA-CuNC fluorescence. PPi is formed by enzymatic action of PPasei.

Erythrocyte Inosine triphosphatase activity: A potential biomarker for adverse events during combination antiretroviral therapy for HIV.

The purine analogues tenofovir and abacavir are precursors of potential substrates for the enzyme Inosine 5'-triphosphate pyrophosphohydrolase (ITPase). Here, we investigated the association of ITPase activity and ITPA genotype with the occurrence of adverse events (AEs) during combination antiretroviral therapy (cART) for human immunodeficiency virus (HIV) infection. In 393 adult HIV-seropositive patients, AEs were defined as events that led to stop of cART regimen. ITPase activity ≥4 mmol IMP/mmol Hb/hour was considered as normal. ITPA genotype was determined by testing two ITPA polymorphisms: c.94C>A (p.Pro32Thr, rs1127354) and c.124+21A>C (rs7270101). Logistic regression analysis determined odds ratios for developing AEs. In tenofovir-containing regimens decreased ITPase activity was associated with less AEs (p = 0.01) and longer regimen duration (p = 0.001). In contrast, in abacavir-containing regimens decreased ITPase activity was associated with more AEs (crude p = 0.02) and increased switching of medication due to AEs (p = 0.03). ITPA genotype wt/wt was significantly associated with an increase in the occurrence of AEs in tenofovir-containing regimens. Decreased ITPase activity seems to be protective against occurrence of AEs in tenofovir-containing cART, while it is associated with an increase in AEs in abacavir-containing regimens.

Ectonucleotidase and adenosine deaminase as inflammatory marker in dairy cows naturally infected by Dictyocaulus viviparus.

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 (P<0.05) 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 (P>0.05). 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.

Variant Inosine Triphosphatase Phenotypes Are Associated With Increased Ribavirin Triphosphate Levels.

Individuals with lower inosine triphosphatase (ITPA) enzyme activity have a reduced likelihood of experiencing hemolytic anemia during hepatitis C virus (HCV) treatment containing ribavirin (RBV). Because ITPA degrades purines and RBV is a purine analogue, it is conceivable that ITPA activity may affect intracellular RBV concentrations. Here we assessed the association between ITPA activity phenotype and concentrations of RBV triphosphate (RBV-TP) in red blood cells (RBCs) during HCV treatment. RBV-TP was quantified in the RBCs of 177 HCV-infected individuals at a median (range) of 84 (19 to 336) days into HCV treatment that included RBV. Mean (SD) RBV-TP concentrations were 92.8 (51.6), 101.3 (53.5), 184.8 (84.5), and 197.7 (64.6) pmol/106 cells for 100%, 60%, 30%, and ≤10% ITPA activity groups, respectively. Overall, RBV-TP was approximately 2-fold higher in patients with ≤30% ITPA activity compared to 100% activity (P < .0001). Despite higher RBV-TP levels, individuals with variant ITPA phenotypes had less anemia. The 100% activity group had, on average, a -2.20 g/dL drop in hemoglobin vs -1.43 g/dL (P = .04) for 60% activity, -1.14 g/dL (P = .008) for 30% activity, and -0.70 g/dL (P = .06) for ≤10% activity. This finding of higher RBV-TP concentrations in RBCs in ITPA variants was unexpected given that ITPA activity-deficient individuals have a reduced likelihood of RBV-induced anemia. It also refutes the hypothesis that the mechanism by which ITPA variants are protected against anemia is due to lower RBV-TP levels in RBCs.

Memory deficit, toxic effects and activity of Na(+), K(+)-ATPase and NTPDase in brain of Wistar rats submitted to orally treatment with alpha-terpinene.

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<0.05) in rats treated with all doses of α-terpinene. The evaluation of DNA damage in brain tissue revealed an increase (p<0.05) on frequency of damage and damage index in all concentrations. According to the cytotoxicity assay, doses of 0.5, 0.75 and 1.0mLkg(-1) increase the lactate dehydrogenase levels, and doses of 1.0mLkg(-1) also decrease (p<0.05) cell viability in brain cells. A decrease (p<0.05) on Na(+), K(+)-ATPase activity in brain tissue and on NTPDase activity in serum were observed in all concentrations of α-terpinene. These results suggest that the α-terpinene was cytotoxic and genotoxic to the brain cells by inducing loss of cell viability and DNA damage, as well as causing alterations in Na(+), K(+)-ATPase and NTPDase activity, what may contribute to the memory deficit of treated animals. Thus, α-terpinene cannot be consumed by the population at the doses studied.

Seric and hepatic NTPDase and 5' nucleotidase activities of rats experimentally infected by Fasciola hepatica.

The enzymatic activities of NTPDase and 5'nucleotidase are important to regulate the concentration of adenine nucleotides, known molecules involved in many physiological functions. Therefore, the objective of this study was to evaluate the activity of NTPDase and 5'nucleotidase in serum and liver tissue of rats infected by Fasciola hepatica. Rats were divided into two groups: uninfected control and infected. NTPDase activity for adenosine triphosphate (ATP) and ADP substrates in the liver was higher compared with the control group at 15 days post-infection (PI), while seric activity was lower. In addition, seric and hepatic samples did not show changes for 5'nucleotidase activity at this time. On the other hand, either NTPDase or 5'nucleotidase activities in liver homogenate and serum were higher at 87 days PI. Early in the infection, low NTPDase activity maintains an increase of ATP in the bloodstream in order to activate host immune response, while in hepatic tissue it decreases extracellular ATP to maintain a low inflammatory response in the tissue. As stated, higher NTPDase and 5'nucleotidase activities 87 days after infection in serum and tissue, probably results on an increased concentration of adenosine molecule which stimulates a Th2 immune response. Thus, it is possible to conclude that F. hepatica infections lead to different levels of nucleotide degradation when considering the two stages of infection studied, which influences the inflammatory and pathological processes developed by the purinergic system.

Role of ITPA and IL28B variants in the management of chronic hepatitis C treatment.

The inosine triphosphatase (ITPA) gene and interleukin 28B (IL28-B) gene variants have been associated to protection of anemia and sustained virological response, respectively, in patients with chronic hepatitis C (CHC) during antiviral therapy. Aim of this study was to evaluate the single and combined role of both polymorphisms in the management of peg-interferon-ribavirin treatment in CHC patients. We studied 79 Italian patients with histology proven CHC treated with pegylated interferon plus ribavirin for 6-12 months on the base of HCV genotype. Patients were carefully followed-up for anemia development which was classified as mild, moderate or severe in relation to levels of haemoglobin decreasing; ribavirin dosage reduction and/or epoietin administration were carried out, where needed. Sustained virological response (SVR) was considered for HCV-RNA clearance after 6 months of treatment stop. Decay of haemoglobin at month 1 of treatment significantly correlated with ITPA activity (p 0.0004) and at multivariate analysis ITPA activity was the only parameter associate with anemia (R - 0.4; p 0.0004). SVR was obtained in 47% of patients. IL28B CC variant was associated with SVR (p 0.01), but IL28B polymorphisms had no influence on the ITPA polymorphism. This study confirms the role of ITPA variants in the prediction of development of severe anemia during antiviral treatment for CHC and demonstrates the absence of influence of IL28B variant on ITPA polymorphisms. These two polymorphisms can be useful in the management of patients that need antiviral therapy for HCV chronic infection.

A disease marker for aspirin-induced chronic urticaria.

There are currently no diagnostic methods in vitro for aspirin-induced chronic urticaria (AICU) except for the provocation test in vivo. To identify disease markers for AICU, we investigated the single nucleotide polymorphism (SNP) of the promoter loci of high-affinity IgE receptor (FcεRIα) and CD203c expression level in Chinese patients with AICU. We studied two genotypic and allelic frequencies of rs2427827 (-344C/T) and rs2251746 (-66T/C) gene polymorphisms of FcεRIα in 20 patients with AICU, 52 subjects with airway hypersensitivity without aspirin intolerance, and 50 controls in a Chinese population. The results showed that the frequencies of two SNPs (-344C>T, -66C>T) were similar to the normal controls. The allele frequency of -344CC was significantly higher in the patients with AICU compared to those with airway sensitivity (p=0.019). We also studied both histamine release and CD203c expression on KU812 cells to assess aspirin-induced basophil activation. We found that the activity of basophil activation of AICU was significantly higher in the patients with AICU compared to those with airway hypersensitivity without aspirin intolerance. The mean fluorescence intensity of the CD203c expression were 122.5±5.2 vs. 103.3±3.3 respectively, (p<0.05), and the percentages of histamine release were 31.3%±7.4% vs. -24.0%±17.5%, (p<0.05) respectively. Although the mean fluorescence intensity of CD203c expression and the percentage of histamine release were significantly up-regulated by aspirin, they were not affected by anti-IgE antibodies. These results suggest that a single SNP of FcεRIα (-344C>T) is less likely to develop AICU and the basophil activation activity in the sera by measuring CD203c expression can be applicable to confirm the diagnosis of AICU.

Activation of basophils is a new and sensitive marker of biocompatibility in hemodialysis.

The hemodialysis procedure involves contact between peripheral blood and the surface of dialyzer membranes, which may lead to alterations in the pathways of innate and adaptive immunity. We aimed to study the effect of blood-membrane interaction on human peripheral basophils and neutrophils in hemodialysis with high- and low-permeability polysulfone dialyzers. The surface expression of CD203c (basophil selection marker) and CD63 (activation marker) after activation by the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fMLP) or anti-Fcε receptor I (FcεRI) antibody and the absolute number of basophils was investigated before and after hemodialysis with each of the dialyzers. Moreover, the expression on neutrophils of CD11b, the CD11b active epitope, and CD88 was analyzed in the same groups of individuals. The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared with that in healthy controls, but no differences were observed after activation by anti-FcεRI. During the hemodialysis procedure, the low-flux membrane induced up-regulation of CD63 expression on basophils, while passage through the high-flux membrane did not significantly alter the responsiveness. In addition, the absolute number of basophils was unchanged after hemodialysis with either of the dialyzers and compared with healthy controls. We found no significant differences in the expression of the neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88) comparing the two different dialyzers before and after dialysis and healthy controls. Together, these findings suggest that alterations in basophil activity may be a useful marker of membrane bioincompatibility in hemodialysis.

The pharmacokinetic effect of adalimumab on thiopurine metabolism in Crohn's disease patients.

BACKGROUND AND AIMS: A drug interaction between infliximab and azathioprine has previously been reported in Crohn's disease patients: the concentration of the main active thiopurine metabolites, the 6-thioguanine nucleotides (6-TGN), increased 1-3 weeks after the first infliximab infusion by 50% compared to baseline. The aim of this prospective study was to determine the effect of adalimumab on thiopurine metabolism in Crohn's disease patients, evaluated by 6-TGN and 6-methylmercaptopurine ribonucleotides (6-MMPR) concentration measurement. METHODS: Crohn's disease patients on azathioprine or mercaptopurine maintenance therapy starting with concomitant adalimumab treatment were included. 6-TGN and 6-MMPR concentrations were determined before initiation of adalimumab and after 2, 4, 6 and 12 weeks of combination therapy. The activity of three essential enzymes involving thiopurine metabolism, thiopurine S-methyltransferase (TPMT), hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and inosine-triphosphate pyrophosphatase (ITPase), was evaluated at baseline and week 4. Clinical outcome was evaluated by the Crohn's disease activity index and C-reactive protein concentrations at baseline, week 4 and week 12. RESULTS: Twelve Crohn's disease patients were analyzed. During the follow-up period of 12 weeks the median 6-TGN and 6-MMPR concentrations did not significantly change compared to baseline. TPMT, ITPase and HGPRT enzyme activity did not change either after 4 weeks. In two patients (17%) myelotoxicity was observed within 2-4 weeks, in whom both low therapeutic 6-TGN and 6-MMPR concentrations were found. CONCLUSIONS: In this study in Crohn's disease patients no pharmacokinetic interaction was shown between adalimumab and the conventional thiopurines, azathioprine and mercaptopurine.

Inhibition of allergen-induced basophil activation by ASM-024, a nicotinic receptor ligand.

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) were identified on eosinophils and shown to regulate inflammatory responses, but nAChR expression on basophils has not been explored yet. OBJECTIVE: We investigated surface receptor expression of nAChR α4, α7 and α1/α3/α5 subunits on basophils. Furthermore, we examined the effects of ASM-024, a synthetic nicotinic ligand, on in vitro anti-IgE and in vivo allergen-induced basophil activation. METHODS: Basophils were enriched from the peripheral blood of allergic donors and the expression of nAChR subunits and muscarinic receptors was determined. Purified basophils were stimulated with anti-IgE in the presence of ASM-024 with or without muscarinic or nicotinic antagonists for the measurement of CD203c expression and histamine release. The effect of 9 days of treatment with 50 and 200 mg ASM-024 on basophil CD203c expression was examined in the blood of mild allergic asthmatics before and after allergen inhalation challenge. RESULTS: nAChR α4, α7 and α1/α3/α5 receptor subunit expression was detected on basophils. Stimulation of basophils with anti-IgE increased CD203c expression and histamine release, which was inhibited by ASM-024 (10(-5) to 10(-)(3) M, p < 0.05). The effect of ASM-024 was reversed in the presence of muscarinic and nicotinic antagonists. In subjects with mild asthma, ASM-024 inhalation significantly inhibited basophil CD203c expression measured 24 h after allergen challenge (p = 0.03). CONCLUSION: This study shows that ASM-024 inhibits IgE- and allergen-induced basophil activation through both nicotinic and muscarinic receptors, and suggests that ASM-024 may be an efficacious agent for modulating allergic asthma responses.

E-NTPDase and E-ADA activities in lymphocytes associated with the immune response of rats experimentally infected with Toxoplasma gondii.

An investigation of E-NTPDase and E-ADA activities in lymphocytes from rats experimentally infected with Toxoplasma gondii was carried out in this study. For this purpose, twenty four adult male Wistar rats were divided in two groups/four subgroups (A1 and A2; B1 and B2-6 animal/each group), with "A" as uninfected and "B" inoculated with T. gondii (RH strain). Sampling was performed on days 5 and 10 post-infection (p.i.), with evaluation of hemogram, immunoglobulins (IgM and IgG) and activity of E-NTPDase and E-ADA in lymphocytes. Enzymes essays showed ATP hydrolysis increased on days 5 (P<0.05) and 10 (P<0.01) p.i., as well as an increase of ADP hydrolysis on day 10 (P<0.01) p.i. E-ADA activity on lymphocytes was also increased in both evaluated periods (P<0.01). Based on E-NTPDase and E-ADA increased activities observed on lymphocytes, it is possible to suggest their involvement in an anti-inflammatory response, consisting of a modulatory response, preventing excessive tissue damage caused by the infection with Toxoplasma gondii.

Loss of ectonucleotidases from the coronary vascular bed after ischemia-reperfusion in isolated rat heart.

BACKGROUND: Ectonucleotidase plays an important role in the regulation of cardiac function by controlling extracellular levels of adenine nucleotides and adenosine. To determine the influence of ischemia-reperfusion injury on ectonucleotidase activity in coronary vascular bed, we compared the metabolic profile of adenine nucleotides during the coronary circulation in pre- and post-ischemic heart. METHODS: Langendorff-perfused rat hearts were used to assess the intracoronary metabolism of adenine nucleotides. The effects of ischemia on the adenine nucleotide metabolism were examined after 30 min of ischemia and 30 min of reperfusion. Adenine nucleotide metabolites were measured by high performance liquid chromatography. RESULTS: ATP, ADP and AMP were rapidly metabolized to adenosine and inosine during the coronary circulation. After ischemia, ectonucleotidase activity of the coronary vascular bed was significantly decreased. In addition, the perfusate from the ischemic heart contained a considerable amount of enzymes degrading ATP, AMP and adenosine. Immunoblot analysis revealed that the perfusate from the ischemic heart dominantly contained ectonucleoside triphosphate diphosphohydrolase 1, and, to a lesser extent, ecto-5'-nucleotidase. The leakage of nucleotide metabolizing enzymes from the coronary vascular bed by ischemia-reperfusion was more remarkable in aged rats, in which post-ischemic cardiac dysfunction was more serious. CONCLUSION: Ectonucleotidases were liberated from the coronary vascular bed by ischemia-reperfusion, resulting in an overall decrease in ectonucleotidase activity in the post-ischemic coronary vascular bed. These results suggest that decreased ectonucleotidase activity by ischemia may exacerbate subsequent reperfusion injury, and that levels of circulating ectonucleotidase may reflect the severity of ischemic vascular injury.

Ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) activities in prostate cancer patients: influence of Gleason score, treatment and bone metastasis.

The relation between adenine nucleotides and cancer has already been described in literature. Considering that the enzymes ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) act together to control nucleotide levels, we aimed to investigate the role of these enzymes in prostate cancer (PCa). E-NPP and ADA activities were determined in serum and platelets of PCa patients and controls. We also verified the influence of the Gleason score, bone metastasis and treatment in the enzyme activities. Platelets and serum E-NPP activity increased, whereas ADA activity in serum decreased in PCa patients. In addition, Gleason score, metastasis and treatment influenced E-NPP and ADA activities. We may propose that E-NPP and ADA are involved in the development of PCa. Moreover, E-NPP and ADA activities are modified in PCa patients with distinct Gleason score, with bone metastasis, as well as in patients under treatment.

Evidence for the role of Epstein Barr Virus infections in the pathogenesis of acute coronary events.

BACKGROUND: The role of viral infections in the pathogenesis of atherosclerosis remains controversial largely due to inconsistent detection of the virus in atherosclerotic lesions. However, viral infections elicit a pro-inflammatory cascade known to be atherogenic and to precipitate acute ischemic events. We have published in vitro data that provide the foundation for a mechanism that reconciles these conflicting observations. To determine the relation between an early viral protein, deoxyuridine triphosphate nucleotidohydrolase (dUTPase), produced following reactivation of Epstein Barr Virus (EBV) to circulating pro-inflammatory cytokines, intercellular adhesion molecule-1 (ICAM-1) and acute coronary events. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples were obtained from 299 patients undergoing percutaneous coronary intervention for stable angina (SA), unstable angina (UA), or acute myocardial infarction (AMI). Plasma concentrations of pro-inflammatory cytokines and neutralizing antibody against EBV-encoded dUTPase were compared in the three patient groups. AMI was associated with the highest measures of interleukin-6 (ANOVA p<0.05; 4.6 ± 2.6 pg/mL in patients with AMI vs. 3.2 ± 2.3 pg/mL in SA). ICAM-1 was significantly higher in patients with AMI (ANOVA p<0.05; 304 ± 116 pg/mL in AMI vs. 265 ± 86 pg/mL SA). The highest values of ICAM-1 were found in patients having an AMI and who were antibody positive for dUTPase (ANOVA p=0.008; 369 ± 183 pg/mL in AMI and positive for dUTPase vs. 249 ± 70 pg/mL in SA negative for dUTPase antibody). CONCLUSIONS/SIGNIFICANCE: These clinical data support a model, based on in vitro studies, by which EBV may precipitate AMI even under conditions of low viral load through the pro-inflammatory action of the early protein dUTPase that is produced even during incomplete viral replication. They further support the putative role of viral infections in the pathogenesis of atherosclerosis and coronary artery events.