In vivo models for heart failure research.

The medical treatment of heart failure (HF) is associated with 50% survival at 5 years, thus being one of the major causes of mortality in Western countries. An understanding of the pathophysiology of HF is essential for the development of novel efficient therapies. Consequently, the use of animal models is indispensable. In addition, the development of new in vivo models of HF is critical for the evaluation of treatments such as gene therapy, mechanical devices and new surgical approaches. However, every animal model has advantages and limitations and none of them is suitable to study all aspects of HF. Besides the technical determinants of a model, species, strain and gender affect the pathophysiology of a given heart pathogenesis and, therefore, have to be considered in each animal model. The most common in vivo models used in cardiology research and in particular in HF remodeling are presented.

Tetranectin expression in gastric adenocarcinomas.

AIMS: The aim was to analyze the immunohistochemical localization of tetranectin in gastric adenocarcinomas and the adjacent tissues of the wall of the stomach. METHODS AND RESULTS: Forty cases of gastric adenocarcinomas were stained by the indirect immunoperoxidase method. Of the ten cases of mucinous signet ring cell carcinomas 5 showed high, 3 moderate and 2 low tetranectin expression. Of the ten cases of well-differentiated intestinal type adenocarcinomas (ITA) 4 showed moderate regional, 3 low regional and 3 negative tetranectin expression. Of the ten cases of moderately-differentiated ITA 3 showed moderate regional, 4 low regional and 3 negative tetranectin expression. Of the ten cases of poorly-differentiated ITA 4 showed focal low and 6 negative tetranectin expression. Overall, the mucinous signet ring carcinomas showed significantly higher tetranectin expression compared to ITA (chi2 = 3.95, p<0.05). In contrast, no significant relationship was found between tetranectin expression and the degree of differentiation in ITA (chi2 = 2.5, p>0.05). In all cases, the perineoplastic desmoplastic reactive stroma showed high expression of tetranectin intra- and extracellularly. The mast cells and goblet cells in the areas of intestinal metaplasia showed high tetranectin expression. CONCLUSIONS: This study shows that: a) tetranectin is produced and deposited extracellularly in the desmoplastic peritumoral stroma of infiltrating gastric adenocarcinomas; b) tetranectin is more highly expressed by the mucinous signet ring cell carcinomas compared to ITA; and c) the amount of tetranectin produced by the ITA is unrelated with the degree of tumor differentiation.

Modulation of rat brain synaptosomal plasma membrane achieved by atracurium and its metabolite laudanosine.

OBJECTIVE: Atracurium besylate and laudanosine cause excitement and seizures when introduced into the central nervous system of laboratory animals. We examined the modulation of lipid-protein interaction in the lipid environment of rat brain synaptosomal plasma membrane (SPM)-bound enzymes as a possible mechanism leading to these effects. METHODS: The effect of various concentrations of atracurium besylate and laudanosine, or of varying duration of SPM, on the activity of Na+/K+-stimulated ATPase, Mg2+-stimulated ATPase and 5'-nucleotidase were assessed. The modulation of lipid protein interaction by laudanosine was estimated on the basis of the temperature dependence and cooperative behaviour of Na+/K+-stimulated ATPase. RESULTS: The effect of atracurium besylate or laudanosine on Na+/K+-stimulated ATPase activity was biphasic. Maximal enzyme stimulation appeared at 10(-4) M atracurium besylate or 10(-8) M laudanosine, and at 30 min of pre-incubation with both drugs. Arrhenius plots of Na+/K+-stimulated ATPase showed a transition temperature of 23.0 +/- 1.2 degrees C in control SPM and shifted to 16.5 +/- 0.9 degrees C (p < 0.01) in SPM treated with 10(-8) M laudanosine. The Hill coefficients for the allosteric inhibition of Na+/K+-stimulated ATPase by fluoride decreased from 1.99 +/- 0.22 in controls to 1.06 +/- 0.11 (p < 0.001) in the presence of 10(-8) M laudanosine. CONCLUSIONS: Our results suggest that laudanosine, one of the principal metabolites of atracurium besylate, affects nerve cell function in rats through the perturbation of the membrane lipid structure accompanied by SPM-bound enzyme dysfunction.

A New Mononuclear Cell (MNC) RNase H Activity-Based Parameter (ψ) With Possible Prognostic Value in Assessing Progression in Acute Myeloid Leukaemia.

A new biological parameter (ψ) has been obtained and proposed here to serve in the assessment of acute myeloid leukemia (AML) progression. It is a function of the activity of RNase H (EC 3.1.4.34), the latter determined in mononuclear cells from the peripheral blood of AML patients. Using a series of patients at the time of diagnosis and after 1-2 cycles of chemotherapy, the enzyme was assayed before the several times during chemotherapy. The derivation of 4 was based on evidence suggesting that the enzyme level correlates with the proliferating leukaemic blasts and their progenitors. Values ψ>1 signify the presence of clonogenic leukaemic progenitor cells in the peripheral circulation. When these high (> 1) ψ values were found during chemotherapy, in these cases it was possible to predict an increase of the peripheral blast pool, with 82% success, occurring 5-35 days before cytologic relapse. In the patients in whom at some stage during treatment, ψ acquired values above unity or in whom ψ increased progressively, survival time was in linear correlation with the time period from the initiation of treatment to the documentation of this high ψ estimate. These results suggest that a patient's relapse risk can be defined by ψ with some degree of precision.

Comparative study of serum and synovial fluid interleukin-11 levels in patients with various arthritides.

OBJECTIVE: To determine the levels of serum and synovial fluid (SF) interleukin (IL)-11 in patients with various arthritides and estimate the contribution of IL-11 to acute phase response (APR). DESIGN AND METHODS: Serum and SF IL-11 were measured by ELISA in patients with rheumatoid arthritis (RA, n = 31), seronegative spondyloarthritis (SSA, n = 23), gout (GT, n = 14) and osteoarthritis (OA, n = 20) and were correlated with ESR and acute phase proteins as well as with cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF alpha. RESULTS: IL-11 was detected in both serum and SF in each group, with IL-11 being statistically higher in SF than serum in all groups, suggesting reduced catabolism or increased synthesis of IL-11 intra-articularly. Median SF IL-11 levels were higher in OA patients than in other groups and in the treated than in the untreated RA subgroup. Moreover, serum and SF IL-11 were correlated significantly with each other, and moderately with the other cytokines examined in RA, SSA, and GT, but not in OA patients, while a significant negative correlation was found with a few of the inflammatory markers examined in each group. CONCLUSIONS: Our findings provide evidence of extensive intra-articular expression of IL-11 in arthritides, especially in OA and treated RA patients, suggesting a protective role for IL-11 in joints, probably through the induction of tissue inhibitor of metalloproteinases.

Serum levels of tetranectin, intercellular adhesion molecule-1 and interleukin-10 in B-chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVE: The fibrinolytic regulator tetranectin (TN), in association with the circulating intercellular adhesive molecule-1 (cICAM-1) and interleukin -10 (IL-10), may be involved in the metastatic cascade of B-chronic lymphocytic leukemia (B-CLL). Our aim was to investigate the potential usefulness of these molecules as prognostic markers in B-CLL. DESIGN AND METHODS: Therefore, TN, cICAM-1, and IL-10 were assessed (ELISA) in the serum of 53 B-CLL patients, classified in Binet A, B, and C stages in comparison with those in 45 healthy subjects (HS). RESULTS: TN was significantly lower in B-CLL patients than in HS (9.63 [8.75-11.51] mg/L, 13.75 [12.56-14.64] ng/mL, respectively, p<10(-5)), being lower (p = 0.05) in B and C stage patients (subgroup B+C) than in A stage ones (subgroup A). cICAM-1 levels were significantly higher in B-CLL patients than in HS (475.86 [355.86-593.79] ng/mL vs. 225.62 [118.49-312.83] ng/mL, respectively, p<10(-5)) with a tendency for higher levels in subgroup B+C than in subgroup A. A significant correlation of cICAM-1 with lactate dehydrogenase (LDH) (r(s) = 0.532, p = 0.049), and a significant increase in cICAM-1 in B-CLL with diffuse bone marrow infiltration (BMI) compared to that in B-CLL with nondiffuse BMI (624.48 [557.24-726.55] ng/mL vs. 480.34 [368.96-590.34] ng/mL, respectively, p = 0.0172) were found. A significant negative correlation between TN and cICAM-1 (r = -0.5017, p = 0.0001) was observed. IL-10 was detected in all B-CLL patients and in no HS (7.37 [5.30-10.55] pg/mL), being higher (p = 0.0153) in C than in A stage patients. A significant correlation of IL-10 with TN and cICAM-1 in subgroup B+C (r(s) = -0.659 [p = 0.014] and r = 0.679 [p = 0.011], respectively) was found. CONCLUSIONS: The abovementioned findings and good performance characteristics of TN and cICAM-1 in B-CLL suggest the potential usefulness of these adhesive/recognition molecules as prognostic markers in B-CLL. The implication of these molecules along with IL-10 in the disease process deserves further study.

Time course of cardiac conditioned responses in restrained rats as a function of the trace CS-US interval.

Two experiments aimed at understanding the temporal characteristics of trace-conditioned heart-rate responses to a 0.5-s tone (conditioned stimulus [CS]) in restrained rats. A CS paired with a tail-shock (unconditioned stimulus [US]) elicited lasting bradycardiac responses. The magnitude and extinction rate of conditioned responses (CR) were independent of the CS-US interval (interstimulus interval [ISI], 3 s to 20 s). Unreinforced test trials were analyzed for CR topography. Responding was delayed in groups with longer ISIs, but CR latencies, peak and decay times were not proportional to the ISI. Response peaks tended to cluster either about 6 s after CS onset, or about 10 s with a slow decay, depending on the ISI. The authors postulated 2 components of auditory stimulus traces involved in cardiac conditioning, maximally active 6 s and 10 s respectively after CS onset. The topography of the CR could be constrained to combinations of associative strength and instantaneous activity of these 2 components.

RNase H of human leukemic cells: a new biological parameter in the study of human leukemias (review).

This article reviews the authors' investigation of the enzyme RNase H (EC 3.1.4.34.) in human leukemic cells and presents the accumulated available data, based on which this enzyme is proposed to serve as a new biological parameter in the study of progression of human leukemias. The introduction gives a brief account of the occurrence, characterization and possible biological role of RNase H in cells and in retroviruses. The results reviewed briefly concern: (1) the development of a new convenient, economic and reliable assay for normal and leukemic blood mononuclear cell RNase H, which is capable of resolving subtle activity differences between samples; (2) the differentiation of RNase H levels between normal and leukemic cells; (3) the correlation of RNase H levels from different leukemia types with the severity of the disease; (4) the correlation of RNase H levels in leukemic cells with clonogenic stages in the clonal differentiation pathway; (5) the predictive potential of a RNase H activity-based parameter (phi) in assessing progression in acute myelocytic leukemia and (6) the possibility of differentiation of the RNase H levels between normal and leukemic cells via regulation of the enzyme activity at the level of antagonistic phosphorylations mediated by cAMP and calmodulin.

Tetranectin levels in patients with acute myocardial infarction and their alterations during thrombolytic treatment.

Tetranectin (TN), a new regulator of fibrinolysis, was studied in the plasma of 60 patients with acute myocardial infarction (AMI) and 30 healthy subjects (HS), in relation to D-dimer (DD) and alpha 2-plasmin inhibitor (alpha 2-PI), to investigate its possible involvement in the pathophysiology of AMI. Thirty patients underwent thrombolytic treatment with fibrin-specific plasminogen activator (rt-PA) (group A); the other 30 patients, according to the exclusion criteria, were conventionally treated (group B). Twenty of the thrombolysized patients established early recanalization (subgroup A1), while 10 failed to respond to thrombolytic treatment (subgroup A2). Median (interquartile range), baseline plasma TN levels were lower in AMI patients compared to HS [8.27 (2.75) mg/L versus 12.1 (0.55) mg/L, P < 10(-6)]. In subgroup A1, TN increased at the end of rt-PA infusion and returned to the baseline levels 12 h later. A positive association between DD and TN release (3 h level minus baseline level) was found (rs = 0.48, P = 0.03) in subgroup A1. No significant alterations of TN levels were observed during therapy in subgroup A2 and group B. TN, DD and alpha 2-PI concentrations in group B remained relatively constant during the study period. This study provides evidence of a significant decrease of TN levels in AMI patients compared to healthy subjects and of a remarkable difference in the evolution of TN levels during thrombolytic treatment with rt-PA between recanalized and non-recanalized AMI patients. Thus, an involvement of TN in the formation and dissolution of fibrin clot in AMI patients is worthy of further investigation.

Imbalance of tissue-type plasminogen activator (t-PA) and its specific inhibitor (PAI-1) in patients with rheumatoid arthritis associated with disease activity.

The relationship between plasma fibrinogen, D-dimer (DD), t-PA and PAI-1 and their correlation with disease activity (DA) were studied in 45 patients with rheumatoid arthritis (RA) (group B) to further understand the implication of fibrinolysis in the pathophysiology of RA. The control group constituted 24 healthy subjects (group A). A Stoke index (SI) of DA was assigned to each patient. Patients were divided into two groups: C, minimal-mild DA (SI 1-7); D, moderate-severe DA (SI 8-17). Fibrinogen was elevated in RA correlating positively with SI and CRP. Hypercoagulability counteracted by reactive fibrinolysis was inferred from a 10-fold increase of DD in group B as compared to group A. The relatively lower levels of DD in group D compared to group C and their negative correlation with SI (r(s) = -0.49, p = 0.0006) indicate the tendency of fibrinolysis to decrease with the increase of DA. Significant elevation of t-PA and PAI-1 were found in group B compared to group A. While t-PA progressively decreased with the increase of DA (r(s) = -0.45, p = 0.0019), a positive relation of PAI-1 to DA was observed (r = 0.42, p = 0.0042). A 2-fold increase of PAI-1/t-PA molar ratio in group D compared to groups A and C as well as its positive correlation with SI (r(s) = 0.63, p = 0.0001) indicate the displacement of balance between t-PA and PAI-1 in favour of the inhibitor with the increase of DA in RA. The involvement of inflammatory mediators in PAI-1/t-PA imbalance was proposed from the relation of fibrinolytic abnormalities with the activity of systemic inflammatory process.

Comparative study of tetranectin levels in serum and synovial fluid of patients with rheumatoid arthritis, seronegative spondylarthritis and osteoarthritis.

Tetranectin (TN) was assessed in paired synovial fluid (SF) and serum (S) samples from 27 patients with rheumatoid arthritis (RA), 23 with seronegative spondylarthritis (SSA) and 22 with osteoarthritis (OA). RA patients had a stronger correlation between serum and SF TN and a higher SF/S TN ratio than did SSA and OA patients. Moreover, the SF/S TN ratio exceeded 1 in most RA patients but not in SSA and OA patients, indicating the possibility of intra-articular TN synthesis in RA. A strong correlation of serum and SF TN with known inflammatory markers was observed in RA. The TN/proteinase inhibitors (PIs: alpha1-antitrypsin, alpha2-macroglobulin) molar ratio in SF was lower in RA and SSA patients to a statistically significant degree than in OA patients. In RA, in contrast to SSA and OA, this ratio correlated positively with the SF interleukin-8 (IL-8), responsible for neutrophil recruitment and degranulation, and negatively with erythrocyte sedimentation rate, serum C-reactive protein and fibrinogen, known markers of disease activity. In conclusion, patients with RA showed lower serum TN levels, a higher SF/S TN ratio and a lower SF TN/PI molar ratio than did SSA and OA patients, suggesting the implication of TN in the impaired regulation of fibrinolysis associated with the inflammatory process.

CD40 expression and its association with low-grade inflammation in a Greek population of type 1 diabetic juveniles: evidence for differences in CD40 mRNA isoforms expressed by peripheral blood mononuclear cells.

BACKGROUND: CD40 signalling has been associated with the pathogenesis of autoimmune diseases and diseases with low-grade chronic inflammation. OBJECTIVE: To investigate, early in the course of type 1 diabetes (T1DM) patients, the expression of CD40 system components, as well as to explore the association of plasma and urine concentrations of CD40 with known inflammatory markers in T1DM. METHODS: Plasma, urine and peripheral blood mononuclear cells (PBMCs) from 70 T1DM patients without clinically detected chronic complications and 40 healthy controls (HCs) were examined using ELISA, western-blot, semi-quantitative RT-PCR and DNA-sequencing. RESULTS: Patients had significantly higher plasma soluble CD40 (sCD40) levels associated with higher Interleukin-6 (IL-6), matrix metalloproteinase-9 (MMP-9) and CRP levels compared with healthy controls. This difference was also evident between poorly and well-controlled diabetic patients. The elevated plasma sCD40 levels do not appear to be due to diminished renal excretion since sCD40 concentrations in the urine were also elevated, suggesting an increased CD40 production. An upregulation of PBMCs' CD40 was evident in T1DM patients associated with higher sCD40, IL-6 and CRP levels. Furthermore, the main CD40 isoform (isoform-I) was solely expressed in poorly controlled diabetics' PBMCs, who also demonstrated cellular CD40 upregulation, higher plasma CD40, CRP, IL-6 and MMP-9 levels compared with the well-controlled diabetics and the control group, who co-expressed type I and II isoforms. CONCLUSIONS: Homeostatic dysregulation of CD40 and its association with inflammatory markers in T1DM patients, especially in those with poor glycaemic control, implies a pathophysiological role of CD40 in the low-grade inflammatory process in T1DM.

Sex differences in oxidant/antioxidant balance under a chronic mild stress regime.

The deterioration of homeostasis between oxidant/antioxidant species may represent an important mechanism linking psychological stress to cardiovascular risk despite the many sex differences in stress responsiveness. The goal of the present study was to investigate the influence of chronic mild stress (CMS), a widely accepted animal model of depression, on oxidative homeostasis-allostasis markers and sICAM-1, a marker of endothelial injury, in the serum of Wistar rats, by taking into account the effect of sex. After six weeks of exposure to mild unpredictable environmental stressors, both male and female rat groups displayed typical changes in hedonic status (anhedonia), which is a core symptom of human depression. Control female rats had higher (nitrite and nitrate) NOx, lower malondealdehyde (MDA) levels with lower activity of antioxidant enzymes and sICAM-1 levels than did control males. CMS induced oxidant/antioxidant responses in both sexes. Females tended to increase their nitric oxide (NO) levels further, while MDA levels did not reach those of males, thus retaining significantly higher NO bioavailability than in males. Concerning the antioxidant enzymes, CMS-females exhibited significantly higher glutathione peroxidase (GPx) activity and lower glutathione reductase (GR) and superoxide dismutase (SOD) activity compared to CMS-males. The CMS response in females was accompanied by lower sICAM-1 levels than in males, suggesting lower endothelial injury. In conclusion, the results of the present study showed that CMS induces different oxidative stress and compensatory responses in both sexes probably due to differences in the mechanisms regulating oxidant/antioxidant pathways.

Assay of hybrid ribonuclease using a membrane filter-immobilized synthetic hybrid: application to the human leukemic cell.

A method for assaying hybrid ribonuclease has been devised which utilizes as substrate the synthetic hybrid [3H]polyriboadenylic acid [poly(rA)]:polydeoxythymidylic acid [poly(dT)] immobilized on the solid matrix of nitrocellulose filters. The hybridization on filter of [3H]poly(rA) to poly(dT) has been explored in terms of efficacy of the process and the response of the product to RNase H. A pulse of uv irradiation of poly(dT) while in dry state on the filter increased its firm binding to the filter in a concentration-dependent manner, resulting in a concomitant increase of the yield of hybrid formation. The filter-immobilized hybrid was 95% resistant to RNase A but sensitive to RNase H. When stored in toluene in the cold the hybrid maintained its stability for over 6 months, as judged by its resistance to RNase A. The method offers a number of advantages over assays that use solution hybrids as substrates and was readily applicable in the screening of leukemic patients, in the leukocytes of which it has demonstrated increased RNase H levels.

Activity and function of hybrid ribonuclease in cells of acute and chronic myelogenous leukemia.

The activity of hybrid ribonuclease (ribonuclease H) has been determined in mononuclear blood cells (lymphocytes plus monocytes) from 23 normal individuals and cells (pool of immature granulocytes, metamyelocytes and lymphocytes) from 35 untreated acute and chronic myelogenous leukemia cases. It was found that in 86% of the leukemic samples the activity of ribonuclease H was above two standard deviations from the mean activity level drawn for the group of normal samples along the 0-100% substrate hydrolysis scale. The activity of the enzyme in leukemic cells correlated linearly with the DNA-synthesizing activity of the cells in vitro and in the examined CML cases it paralleled the inverse relationship of the incorporation of tritiated thymidine into DNA to the size of the pool of immature granulocytes. In one CML patient who received chemotherapy with Myleran, the activity of ribonuclease H, high at the initiation of drug therapy, was reduced to a normal level at remission, but increased again at the stage of subsequent relapse. These findings indicate that the levels of ribonuclease H in leukemic cells reflect the proliferative activity of the population in the cases of untreated myelogenous leukemias.

Activity of ribonuclease H in cells of chronic B-lymphocytic leukaemia: correlation with clinical stage.

Peripheral blood mononuclear cells (PBMNC) from 23 healthy subjects and 39 patients with B-cell chronic leukaemia (B-CLL) were assayed for ribonuclease H activity using as substrate the filter-immobilized synthetic homopolymer hybrid 3H-poly(rA):poly(dT). In 69% of the leukaemia patients examined enzyme activities were above those estimated in cells from the healthy controls. The mean enzyme levels for the normal and the leukaemic samples group were (per cent substrate hydrolysis): 8.1 +/- 8.9 and 58.7 +/- 40.8, respectively, their difference being statistically highly significant (P less than 0.0001). This result does not represent homogeneity within the cells but is due to a subclass of cells within the leukaemic clone containing the enzyme, thus contributing through pool size fluctuation to the wide variations of enzyme activity observed among the patients. These cells containing high activity could not be identified with either the prolymphocytes or the large lymphocytes. The activity of ribonuclease H in the examined CLL patients correlated with disease stage (Binet) (P = 0.011) and appeared to serve as a sensitive indicator of disease progression when compared with a number of other known prognostic parameters.

Plasma tetranectin levels and disease activity in patients with rheumatoid arthritis.

OBJECTIVE: We investigated alterations of levels of plasma tetranectin, a new regulator of plasminogen activation, in patients with rheumatoid arthritis (RA) in relation to disease activity and other fibrinolytic variables. METHODS: Tetranectin (TN), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1) were quantitatively assessed (ELISA) in plasma of 41 patients with RA and 30 healthy subjects, alpha 2-Antiplasmin activity was assessed by the amidolytic method. Disease activity was determined as a composite Stoke Index, which measures inflammatory processes in RA. Patients were divided into 3 groups according to Stoke Index score of disease activity: A, minimal-mild, 1-7: B. moderate. 8-11: C. severe. 12-17. RESULTS: Plasma TN in patients was significantly lower compared to that of healthy subjects [9.11 (4.97-13.49) mg/l, 12.05 (9.50-13.60) mg/l, median (range), respectively; p = 0.0001]. TN decreases with the increase of disease activity from group to group. A significant negative correlation between TN and Stoke Index. C-reactive protein and erythrocyte sedimentation rate was found (rs = -0.49, p = 0.0012; rs = -0.44, p = 0.0044; rs = -0.37, p = 0.016, respectively). alpha 2-Antiplasmin activity was elevated in patients compared to healthy subjects [105.0% (53.0-146.0), 70.6% (48.2-124.0), median (range), respectively; p = 0.0001], showing a negative correlation with Stoke Index (rs = -0.38, p = 0.0139). The close positive correlation of TN with alpha 2-antiplasmin (rs = 0.66, p = 0.0001) and the absence of correlation with t-PA and PAI-1 were explained by the involvement of TN and alpha 2-antiplasmin in localized rather than in systemic fibrinolysis. CONCLUSION: Our findings suggest TN plays a role in the pathophysiology of RA and point to the usefulness of TN assessment as a specific fibrinolytic marker in the evaluation of disease activity in patients with RA. The role of TN in the intraarticular regulation of fibrinolysis, important for the expansion of pannus, tissue remodeling and angiogenesis, is discussed.

Modulation of synaptosomal plasma membrane-bound enzyme activity through the perturbation of plasma membrane lipid structure by bupivacaine.

UNLABELLED: We investigated modulations of lipid dynamics and lipid-protein interactions of rat brain synaptosomal plasma membrane (SPM) as one of the possible mechanisms by which the local anesthetic bupivacaine (BPV) has an adverse effect on nerve cell function, with SPM-bound enzyme activity used as a functional probe. The kinetics of BPV impact on the activity of the endoenzymes Ca2+/Mg2+-stimulated ATPase and Na+/K+-stimulated ATPase and the active concentrations of the drug were relevant to those that produce biphasic systemic toxicity. Arrhenius plots of these enzymes showed a transition temperature of 26.6 +/- 1.8 degrees C and 24.5 +/- 1.2 degrees C (mean +/- SD), respectively, in control SPM, which shifted to 17.1 +/- 0.95 degrees C (P < 0.01) and 18.2 +/- 0.85 degrees C (P < 0.05) in SPM treated with 10(-5) M BPV. The Hill coefficients for the allosteric inhibition of Ca2+/Mg2+-stimulated ATPase by Na+ and Na+/K+-stimulated ATPase by fluoride decreased from 1.73 +/- 0.20 and 1.95 +/- 0.25, respectively, in controls to 0.92 +/- 0.09 (P < 0.001) and 1.09 +/- 0.11 (P < 0.001) in the presence of 10(-5) M BPV. The fluidity perturbation in the microenvironment of the ectoenzyme acetylcholinesterase was observed only at 5 x 10(-3) M BPV, as confirmed by the disparity in transition temperature between the controls (22.3 +/- 1.2 degrees C) and the BPV-treated SPM (17.5 +/- 0.8 degrees C, P < 0.01) and that in the Hill coefficient in the two groups: 2.15 +/- 0.24 and 0.97 +/- 0.12 (P < 0.001), respectively. IMPLICATIONS: We propose that under physiological conditions, the neutral and protonated forms of local anesthetics can affect nerve cell function through the asymmetric perturbation of the membrane lipid structure, accompanied by synaptosomal plasma membrane-bound enzyme dysfunction.

The effect of disease activity related cytokines on the fibrinolytic potential and cICAM-1 expression in rheumatoid arthritis.

OBJECTIVE: We studied the relation of pro and antiinflammatory cytokines to disease activity, coagulation, and fibrinolytic variables as well as to circulating intercellular adhesive molecule- 1 (cICAM-1), so as to better understand the cascade of events implicated in the inflammatory process in rheumatoid arthritis (RA). METHODS: Tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-10, cICAM-1, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor- 1 (PAI-1), and D-dimer antigens were measured by ELISA in the blood of 45 RA patients and 33 healthy subjects (HS). The Stoke Index was used to describe the disease activity in patients, who were divided into subgroups: A: minimal-mild disease activity (n = 23, Stoke Index = 1-7); B: moderate disease activity (n = 12, Stoke Index = 8-11); C: severe disease activity (n = 9, Stoke Index = 12-17). RESULTS: TNF-alpha, IL-6, and IL-10 were significantly higher in RA patients than in HS. TNF-alpha and IL-6, in contrast to IL-10, have the tendency to increase progressively with the increase of disease activity from subgroup to subgroup, correlating significantly with Stoke Index. TNF-alpha and IL-6 correlated positively with PAI-1 and negatively with t-PA and D-dimer. Moreover, a positive correlation of IL-6 with fibrinogen and of both cytokines with PAI-1/t-PA molar ratio were found in all RA patients, while IL-10 showed a significant negative correlation only with PAI-1. Serum cICAM-1 was significantly elevated in RA compared to HS, showing a tendency to increase with the increase of disease activity from subgroup to subgroup. A positive correlation of cICAM-1 with TNF-alpha and IL-6 and a negative one with IL-10 was observed in RA. CONCLUSION: Proinflammatory cytokines TNF-alpha and IL-6 may be implicated in the imbalance of coagulation and fibrinolysis in favor of coagulation and the impairment of the adhesive molecule pathway in RA. This action of TNF-alpha and IL-6 does not seem to be countered by the antiinflammatory cytokine IL-1O action.