[Characteristics of enzymatic state and functional activity of immunocompetent cells in peripheral blood of patients with kidney cancer].

Characteristics of T-cell, humoral component of the immune system, activity of NAD(P)-dependent dehydrogenases in blood lymphocytes, phagocytic activity, and chemiluminescent response of blood neutrophils from patients with renal cell cancer have been examined before and within 14 days after surgical treatment. Changes of these parameters depended on the time elapsed after surgery. It is shown that immune status, enzymatic activity of lymphocytes and functional state of neutrophils in patients with renal cell cancer failed to reach normal levels 14 days after complete nephrectomy.

A new enzymatic cycling method for ammonia assay using NAD synthetase.

BACKGROUND: Ammonia is an important marker for liver diseases such as hepatitis and hepatic cirrhosis. Several methods have been developed for ammonia analysis. In particular, the enzymatic assay using glutamate dehydrogenase has been widely used. However, this method is not necessarily high in sensitivity and accuracy due to inhibition by interferences in plasma and instability over long-term storage. METHODS: We developed an ammonia assay using a system consisting of three enzymes, NAD synthetase (NADS; EC 6.3.1.5), glucose dehydrogenase (GlcDH; EC 1.1.1.47), and diaphorase (DI; EC 1.6.99.2). RESULTS: The calibration curve for ammonia with the cycling method was linear (r=0.999) up to 300 micromol/l. The within-run CVs of 10 and 20 micromol/l NH4Cl solutions and 24.1 micromol/l ammonia in human plasma were 2.3%, 1.5%, and 2.8%, respectively. The between-run CVs of them were 4.5%, 3.1%, and 2.8%, respectively. The recovery was between 96.3% and 105%, and the limit of detection was 2.4 micromol/l. No significant interference was observed with addition of the following components: hemoglobin, bilirubin, chyle, EDTA, heparin, and sodium citrate. Due to the high degree of specificity of NAD synthetase to ammonia, no amino compounds exhibited any effect on the ammonia assay. A high correlation was obtained between results of the present method (y) and a conventional glutamate dehydrogenase method in regression analysis; y=0.944x-6.160 with r=0.993 (n=125). However, an addition error was observed from Bland-Altman analysis (the 95% limits of agreement between the two methods; 9.51+/-5.92 micromol/l). CONCLUSION: This new enzymatic method is more sensitive, precise, and accurate than the conventional method. In particular, accurate assay for ammonia can be performed without interference in the presence of various compounds.

[Correlation of clinical manifestations of atopic dermatitis in children and the activity of NAD(PH)-dependent dehydrogenases in blood lymphocytes]. / Vzaimosviaz' klinicheskikh proiavlenii atopicheskogo dermatita u detei s aktivnost'iu NAD(F)-zavisimykh degidrogenaz limfotsitov krovi.

The bioluminescence was used to study the activity levels of NAD (P)-dependent dehydrogenases in blood lymphocytes of 88 children with atopic dermatitis. The nature and intensity of the investigated enzymes were found to be dependent on age and a disease severity with changes being most pronounced and resistant in the elder age group and in patients with a continuously relapsing disease course. Besides, it was shown that the changes of NAD (P)-dependent dehydrogenases in blood lymphocytes are objectively related with an allergic inflammation clinical course in patients with atopic dermatitis and could be clinically valuable in monitoring the dermatitis progression at the metabolic level, in prognosticating possible relapses and in evaluating a chosen therapy.

Effect of aldosterone and glycyrrhetinic acid on the protein expression of PAI-1 and p22(phox) in human mononuclear leukocytes.

Aldosterone excess can produce heart and kidney fibrosis, which seem to be related to a direct effect of aldosterone at the level of specific receptors. We report a direct, mineralocorticoid-mediated effect on the protein expression of two markers of oxidative stress after incubation of mononuclear leukocytes with 1 x 10(-8) M aldosterone (p22(phox)/beta-actin = 1.38 +/- 0.05 and PAI-1/beta-actin = 1.80 +/- 0.05). The same effect was also found with 3 x 10(-5) M glycyrrhetinic acid, the principal constituent of licorice root (p22(phox)/beta-actin = 1.37 +/- 0.97 and PAI-1/beta-actin = 1.80 +/- 0.04). The effect of both aldosterone and glycyrrhetinic acid is blocked by incubation with added 1 x 10(-6) M of receptor-antagonist canrenone. Canrenone alone did not show any effect. PAI-1 related protein was also found using 4 x 10(-9) M aldosterone. Incubations with 1 x 10(-9) M for 3 hours as well as 1 x 10(-8) M aldosterone for 5, 10, and 20 minutes were ineffective for both proteins. These data support the previous finding of an involvement of mononuclear leukocytes in the pathogenesis of the oxidative stress induced by hyperaldosteronism. In addition, the results confirm our previous data on a direct effect of glycyrrhetinic acid at the level of mineralocorticoid receptors.

[Evaluation of the metabolic state and functional activity of immunocompetent cells in children with relapsing herpetic viral infection]. / Otsenka metabolicheskogo sostoianiia i funktsional'noi aktivnosti immunokompetentnykh kletok u detei s retsidiviruiushchei gerpesvirusnoi infektsiei.

Specific features of the immune response were studied (during the clinical remission period) in 40 children (aged 3 to 6) with a relapsing herpes viral infection (RHVI). The following was established: an increased relative and absolute quantity of lymphocytes, a reduced percentage content of CD+ and an increased absolute count of CD+ in the peripheral blood. Changes in the immunity humoral chain manifested themselves by an increased content of class G immunoglobulins and of circulating immune complexes (CIC) in the blood serum. A reduced quantity of phagocyting blood neutrophils was registered with the absorbing ability being preserved. Changes in the metabolic status of immune-competent cells were established; they were described by a high activity of the plastic exchange reaction, a tensed energetic potential and a reduced glutathione-recovery reaction modulating the proliferative activity of lymphocytes. A reduced reserve potential of oxygen-dependent biocenosis of neutrophyl granulocytes was detected in the peripheral blood. Obviously, the structural and metabolic changes in the blood lymphocytes as well as the abnormalities in the functional activity of neutrophyl granulocytes, observed during the clinical remission period in the RHVI period, are a cause predetermining the relapses of the disease and can be regarded as a criterion for applying the immune rehabilitation.

Divergence of mechanisms regulating respiratory burst in blood and sputum eosinophils and neutrophils from atopic subjects.

Eosinophil respiratory burst is an important event in asthma and related inflammatory disorders. However, little is known concerning activation of the respiratory burst NADPH oxidase in human eosinophils. Conversely, neutrophils are known to assemble NADPH oxidase in intracellular and plasma membranes. We hypothesized that eosinophils and neutrophils translocate NADPH oxidase to distinct intracellular locations, consistent with their respective functions in O(2)(-)-mediated cytotoxicity. PMA-induced O(2)(-) release assayed by cytochrome c was 3.4-fold higher in atopic human eosinophils than in neutrophils, although membrane-permeable dihydrorhodamine-123 showed similar amounts of release. Eosinophil O(2)(-) release was dependent on Rac, in that it was 54% inhibited by Clostridium difficile toxin B (400-800 ng/ml). In eosinophils stimulated with PMA, a pronounced shift of cytosolic Rac to p22(phox)-positive plasma membrane was observed by confocal microscopy, whereas neutrophils directed Rac2 mainly to intracellular sites coexpressing p22(phox). Similarly, ex vivo sputum eosinophils from asthmatic subjects exhibited predominantly plasma membrane-associated immunoreactivity for Rac, whereas sputum neutrophils exhibited cytoplasmic Rac2 staining. Thus, activated sputum eosinophils, rather than neutrophils, may contribute significantly to the pathogenesis of asthma by extracellular release of tissue-damaging O(2)(-). Our findings suggest that the differential modes of NADPH oxidase assembly in these cells may have important implications for oxidant-mediated tissue injury.

[The activity of NAD(P)-dependent dehydrogenases in blood lymphocytes in children and adolescents with different duration of insulin-dependent diabetes mellitus]. / Aktivnost' NAD(P)-zavisimykh degidrogenaz v limfotsitakh krovi u detei i podrostkov s raznoi prodolzhitel'nost'iu insulinozavisimogo diabeta.

The levels of blood lymphocyte NAD(P)-dependent dehydrogenases were investigated in children and teenagers with different duration of insulin-dependent diabetes mellitus (IDDM). The level of some NAD(P)-dependent dehydrogenases changed proportionally in dependence of IDDM duration and insulin therapy did not restore their activities to the normal level. It is suggested that these changes may reflect decrease of energy metabolism and plastic processes in blood lymphocytes from diabetic children and teenagers reflecting. These changes correspond to altered functional reactivity of immunocompetent cells and represent metabolic basis of immunopathogenic complications of IDDM.

[Metabolic characteristics of immunocompetent cells in children with relapsing herpes]. / Osobennosti matabolizma immunokompetentnykh kletok u detei s retsidiviruiushchei gerpeticheskoi infektsiei.

Healthy children aged 1-6 years and patients of the same age with recurrent herpesvirus infection (RHVI) during remission were examined. The levels of NAD(P)-dependent dehydrogenases of blood lymphocytes were measured. Lymphocyte metabolism in the patients was characterized by activation of the key reaction of the pentose phosphate cycle, increased level of the mitochondrial compartment oxidoreductases, increased substrate flow through glucose-3-phosphate dehydrogenase, and inhibition of NADH-dependent dehydrogenase reactions of the cytoplasmatic compartment. A relationship between intracellular metabolism and incidence of RHVI relapses was detected. The metabolic status of blood lymphocytes did not normalize during RHVI remissions.

Rapid detection of polymorphisms of the nitric oxide cascade.

NOS3 (endothelial nitric oxide (NO) synthase) and p22phox (subunit of NAD(P)H oxidase) are two genes whose products are involved in formation and degradation of NO, a ubiquitous signaling molecule largely responsible for the maintenance of normal endothelial function. The G894T polymorphism of NOS3 and the C242T polymorphism of p22phox are reportedly associated with numerous cardiovascular diseases. For each polymorphism we developed a rapid and reliable method with the hybridization probes format on the LightCycler and compared it with conventional PCR-restriction fragment length polymorphism (PCR-RFLP) analysis with regard to reliability, duration and cost. The new methods are more reliable, faster and less expensive than PCR-RFLP analysis and therefore represent a significant advantage in the detection of two candidate risk factors for cardiovascular diseases.

[Clinical significance of NADPH-dependent dehydrogenases in lymphocytes from patients with bronchial asthma]. / Klinicheskaia znachimost' izmenenii aktivnosti NADF-zavisimykh degidrogenaz v limfotsitakh u bol'nykh bronkhial'noi astmoi.

The activities of lymphocyte NADP-dependent dehydrogenases were evaluated in 84 children with atopic asthma during relapses and clinical remission. The pattern and severity of changes in the studied enzymes depended on children's age and duration and severity of disease. Changes in the older age group and during relapse were the deepest and most stubborn. During remission the activities of the studied oxidoreductases did not reach the parameters observed in healthy children. Changes in the activities of NADP-dependent dehydrogenases in blood lymphocytes objectively reflect the course of allergic inflammation in patients with atopic asthma and can be clinically used for evaluation of the pathological process at a metabolic level.

Effects of amitriptyline on diurnal variations of oxidation-reduction enzyme activities in rat lymphocytes during experimental desynchronosis.

We studied the effects of amitriptyline on diurnal variations of oxidative-reduction enzyme activities (succinate dehydrogenase, lactate dehydrogenase, and NADPH diaphorase) in rat peripheral blood lymphocytes during experimental desynchronosis. Desynchronosis was induced by constant light exposure for 14 days and manifested in a morning shift of maximum lactate dehydrogenase activity and elevated morning NADPH diaphorase activity. Amitriptyline normalized morning lactate dehydrogenase activity and diurnal variations in NADPH diaphorase activity.

Transient association of the nicotinamide adenine dinucleotide phosphate oxidase subunits p47phox and p67phox with phagosomes in neutrophils from patients with X-linked chronic granulomatous disease.

Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requires recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized phagocytosis assay and immunofluorescence microscopy (IFM) to examine the association of cytosolic NADPH oxidase subunits with phagosomes containing opsonized zymosan (OpZ). Ingestion of OpZ began within 30 seconds of particle binding and forming phagosomes were enriched for both F-actin and the actin-binding protein p57. NADPH oxidase subunits p47phox and p67phox were also recruited to forming phagosomes and were retained on mature phagosomes for at least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosomes was confirmed by immunoblotting. Translocation of p67phox, but not p57, to forming phagosomes was deficient in PMNs lacking p47phox. Surprisingly, we found that in PMNs from six individuals with X-linked chronic granulomatous disease (CGD), p47phox and p67phox accumulated in the periphagosomal area during ingestion of OpZ. However, in marked contrast to normal PMNs, p47phox and p67phox were shed from nascent phagosomes along with F-actin and p57 once OpZ was internalized (approximately 5 minutes). These data support a model in which flavocytochrome b is required for stable membrane binding of p47phox and p67phox, but not their association with the cytoskeleton or transport to the cell periphery.

2-Hydroxymethyl-1-naphthol diacetate (TAC) suppresses the superoxide anion generation in rat neutrophils.

We have investigated the inhibitory effect of 2-hydroxymethyl-1-naphthol diacetate (TAC) on the respiratory burst of rat neutrophils and the underlying mechanism of action was also assessed in this study. TAC caused concentration-related inhibition of the formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2*-) generation (IC50 10.2+/-2.3 and 14.1+/-2.4 microM, respectively) and O2 consumption (IC50 9.6+/-2.9 and 13.3+/-2.7 microM, respectively) of neutrophils. TAC did not scavenge the generated O2*- during dihydroxyfumaric acid autoxidation. TAC inhibited both the transient elevation of [Ca2+]i in the presence or absence of [Ca2+]o (IC50 75.9+/-8.9 and 84.7+/-7.9 microM, respectively) and the generation of inositol trisphosphate (IP3) (IC50 72.0+/-9.7 microM) in response to fMLP. Cytosolic phospholipase C (PLC) activity was also reduced by TAC at a same range of concentrations. The PMA-induced PKC-beta associated to membrane was attenuated by TAC (about 80% inhibition at 30 microM). Upon exposure to fMLP, the cellular cyclic AMP level was decreased in neutrophils pretreated with TAC. TAC attenuated fMLP-induced phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 (IC50 17.4+/-1.7 microM), but not p38. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP was inhibited by TAC in a concentration-dependent manner (IC50 25.4+/-2.4 and 25.9+/-1.4 microM, respectively). TAC had no effect on the O2*- generation of PMA-stimulated and arachidonic acid (AA)-stimulated NADPH oxidase preparations. However, TAC caused concentration-related decrease of the membrane associated p47phoX in PMA-stimulated neutrophils (about 80% inhibition at 30 microM). We conclude that inhibition by TAC of the neutrophil respiratory burst is probably attributable to the blockade of the p42/44 MAPK and phospholipase D (PLD) pathways, the membrane translocation of PKC, and to the failure in assembly of a functional NADPH oxidase complex. Blockade of the PLC pathway by TAC probably plays a minor role.

Missense mutations in the gp91-phox gene encoding cytochrome b558 in patients with cytochrome b positive and negative X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a disorder of host defense due to genetic defects of the superoxide (O2-) generating NADPH oxidase in phagocytes. A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the oxidase. The X-linked form of the disease is due to defects in the gp91-phox gene. We report here biochemical and genetic analyses of patients with typical and atypical X-linked CGD. Immunoblots showed that neutrophils from one patient had small amounts of p22-phox and gp91-phox and a low level of O2- forming oxidase activity, in contrast to the complete absence of both subunits in two patients with typical CGD. Using polymerase chain reactions (PCR) on cDNA and genomic DNA, we found novel missense mutations of gp91-phox in the two typical patients and a point mutation in the variant CGD, a characteristic common to two other patients with similar variant CGD reported previously. Spectrophotometric analysis of the neutrophils from the variant patient provided evidence for the presence of heme of cytochrome b558. Recently, we reported another variant CGD with similar amounts of both subunits, but without oxidase activity or the heme spectrum. A predicted mutation at amino acid 101 in gp91-phox was also confirmed in this variant CGD by PCR of the genomic DNA. These results on four patients, including those with two variant CGD, are discussed with respect to the missense mutated sites and the heme binding ligands in gp91-phox.

[Neural network classification of people with chronic, nonspecific lung diseases using immunologic parameters of the blood and activity of metabolic enzymes in lymphocytes]. / Neirosetevaia klassifikatsiia lits s khronicheskimi nespetsificheskimi zabolevaniiami legkikh po velichene immunologicheskikh parametrov krovi i aktivnosti metabolicheskikh fermentov limfotsitov.

Informatic importance of blood immunological parameters and activities of lymphocyte metabolic enzymes was investigated in patients with chronic non-specific lung diseases (CNLD) in remission by neural-network classifier. There insignificant increase of immunoregulatory index, absence of variations in indices of humoral immunity, but decrease oxidoreductase activities controlling of bioenergetic processes were detected in immunocompetent cells of patients with CNLD. It was assumed that disturbance of the metabolic state of lymphocytes defined weak functional activation of immunocompetent cells, as well as the development of immunodeficiency and chronicity of diseases. Basing on analysis of lymphocytic NAD(P)-dependent dehydrogenases, neural-network classifier divided all patients with CNLD into two groups distinguishing by the level of intracellular metabolism and the type of immune reactions. This result and complete training on the basis of immunological parameters reflect higher informatic importance of metabolic indices of lymphocytes in diagnosis of immunodeficiency.

Activation of p38 in stimulated human neutrophils: phosphorylation of the oxidase component p47phox by p38 and ERK but not by JNK.

Incubation of human neutrophils with FMLP, a chemotactic peptide, or PMA, a stimulator of protein kinase C, resulted in the activation of p38, a proline-directed kinase. Previous studies had shown that extracellular signal-regulated kinase (ERK), another proline-directed kinase, was activated with similar kinetics in neutrophils stimulated with FMLP and PMA (1, 2). Because one possible target for these proline-directed kinases is p47phox, a component of the respiratory burst oxidase, we examined the phosphorylation of this protein by p38 and ERK, as well as JNK, another proline-directed kinase present in neutrophils. We found that both p38 and ERK phosphorylated p47phox at the same site and at similar rates, but that p47phox was not a substrate for JNK. These data show that p38, like ERK, can be activated in neutrophils exposed to an appropriate stimulus, and that some but not all proline-directed kinases are able to participate in the phosphorylation of a protein essential for normal neutrophil function.

[Neural network modeling of triiodothyronine concentrations by the levels of lymphocytic NADP-dependent dehydrogenases in patients with thyroid hypo- and hyperfunction]. / Neirosetevoe modelirovanie kontsentratsii triiodtironina po urovniam NAD(P)-zavisimykh degidrogenaz limfotsitov krovi u lits s gipo- i giperfunktsiei shchitovidnoi zhelezy.

Whether a computer model which calculates serum triiodothyronine concentrations (T3) can be constructed by the levels of metabolic enzymes in the immunocompetent cells by means of a neural network predictor of real numbers. For this, the concentration of serum T3 and the activity lymphocytic NADP-dependent dehydrogenases were determined by radioimmune assay and bioluminescence technique, respectively. It was found that the neural network prediction might be only in each examined group alone. Conceivably, this is associated with the presence of compensatory processes of intracellular metabolism at high and low concentrations of thyroid hormones. The involvement of the examined enzymes in the metabolic mechanisms of immuno-endocrine interaction both in health and in disease, as well as in the thyroid hypo- and hyperfunction can be judged from the value of the input parameters of the neural network model.

[Activity of NADP-dependent dehydrogenase of thymocytes and blood lymphocytes after irradiation of rabbits in vivo with long-wave laser radiation]. / Aktivnost' HADF-zavisimykh degidrogenaz timotsitov i limfotsitov krovi posle oblucheni krolikov in vivo dlinnovolnovym lazernym islucheniem.

To study the mechanisms of immunocompetent cells' response to long-wave laser radiation in male Chinchilla rabbits after in vivo emission (lambda = 632.8 nm; emissive power, 19 mW) for 15 min, lymphocytes and thymocytes were identified in the projection areas of the thymus and femur and the levels of NADP-dependent dehydrogenases were measured. The changes found in the levels of thymocyte NADP-dependent dehydrogenases while exposing the rabbits to radiation in the area of the femur, as well as the area-related differences in the modulating effects of laser radiation on the activity of the lymphocytic enzymes under study indicate the mediated cellular metabolic regulation which is likely to be chiefly affected by the autonomic nervous system in the first postradiation minutes.

Hyperphosphorylated p47-phox lost the ability to activate NADPH oxidase in guinea pig neutrophils.

p47-phox is one of the cytosolic activation factors of NADPH oxidase in neutrophils and known to translocate to plasma membranes and function by protein kinase C-phosphorylation. In cytosol fraction, prepared from calyculin A-treated neutrophils, the activity of cytosolic factor to activate NADPH oxidase was more reduced than that from PMA-treated cells. But, p47-phox did not translocate to the membranes, even if p47-phox was hyperphosphorylated in the calyculin A-treated neutrophils. Such hyperphosphorylated p47-phox seemed to lose the activity to constitute NADPH oxidase complex.