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.

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.

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.

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.

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.

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.

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.

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.

Calmodulin-related changes in microsomal membrane fluidity during liver regeneration.

Based on our previous findings on the modifying effect of calmodulin (CaM) on the physiochemical properties of biomembrane, we have investigated the possible relationship between intracellular CaM content and endoplasmic reticulum (ER) membrane fluidity and function during liver regeneration. The degree of ER membrane fluidity was estimated by fluorescence polarization analysis with the 1,6-diphenyl-1,3,5-hexatriene probe. Microsomal guanylate cyclase (GC) was used as a functional parameter. The kinetics of the increase in the ER membrane fluidity during liver regeneration was strictly parallel to the CaM surge and was matched by an increase in GC activity. The stimulative effect of splenectomy on liver regeneration and its inhibition by Walker-256 tumor, inferred from the corresponding alterations of CaM levels, were mirrored by the modulation in GC activity. The fluidizing effect of CaM on ER membrane was concluded from the drop in thermotropic transition temperature from 28.3 +/- 1.6 degrees C in control membranes to 17.8 +/- 1.1 degrees C membranes from regenerating livers and to 19.8 +/- 1.2 degrees C in control membranes treated with CaM. Arrhenius plots of GC activity exhibited a transition temperature of 25.5 +/- 1.25 degrees C in controls, which shifted to 20.5 +/- 0.9 degrees C in ER membranes from regenerating livers and to 21.7 +/- 1.1 degrees C in control membranes treated with CaM. The Hill coefficient for the allosteric activation of the GC by Mn.GTP decreased from 1.49 +/- 0.16 in controls to 0.93 +/- 0.085 in membranes from regenerating cells and to 0.86 +/- 0.073 in CaM-treated membranes. Both effects of CaM were consistent with a fluidity increase in the enzyme's lipid microenvironment. The results of the present study suggest that an early key event in liver regeneration may be the CaM-induced modulation of ER membrane fluidity and function.

PGE2 and PGA2 affect the allosteric properties and the activities of calmodulin-dependent guanylate cyclase and Ca2+-stimulated ATPase of Walker-256 tumour microsomal membranes.

PGE2 and PGA2 incubated for 30 min at 25 degrees C with microsomal membranes isolated from Walker-256 tumour, in the presence of 50 microM indomethacin increase the lipid fluidity estimated by steady-state fluorescence anisotropy [(r0/r)-1]-1, using 1,6-diphenyl-1,3,5-hexatriene (DPH) as probe. The microsomal preparations of Walker-256 tumour contained calcium-stimulated and magnesium-dependent ATPase as well as calmoduling-dependent guanylate cyclese activities. A considerable decrease (approx. 65%) in the activity of the Ca2+-stimulated ATPase was observed when preparations were treated with 10 microM PGE2 and PGA2. A dramatic gradual decrease of the calmodulin-dependent guanylate cyclase activity was also observed at different concentrations of PGE2 and PGA2 (0.25-10 microM). The ATP-dependent uptake of calcium was reduced by approximately 60% in microsomal membranes treated with PGE2 and PGA2. The allosteric properties of Ca2+-stimulated ATPase by Na+, and of guanylate cyclase by Mn.GTP (as reflected by changes in the Hill coefficients, h) were modulated by PGE2 and PGA2. The apparent cooperativity of the Ca2+-ATPase (h + 1.73 +/- 0.21) in control membranes was abolished (h + 1.1 +/- 0.11 and h = 0.9 +/- 0.09) in membranes treated by PGE2 and PGA2 (10 microM), while the allosteric stimulation of guanylate cyclase by Mn.GTP was reduced from h = 2.78 +/- 0.24 in control membranes to h = 1.92 +/- 0.16 and h = 1.73 +/- 0.15 in membranes treated by PGE2 and PGA2 (10 microM), respectively, suggesting that the physical state of Ca2+-stimulated ATPase and guanylate cyclase lipid microenvironments changed from a gel phase to a liquid-crystalline phase. In conclusion, it is suggested that PGE2 and PGA2 promote a phase separation in Walker-256 tumour microsomal membranes. This may be relevant to the Ca2+-calmodulin system and tumour growth inhibition.

Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes.

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5-5 microM) follows an exponential curve described by the general formula y = a.ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 microM cholesterol glucoside, with a Hill coefficient of h = 2.98 +/- 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 microM) or its glucoside (up to 50 microM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 microM) incorporation. Cholesterol (5 microM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 +/- 1.1 degrees C in control SPM, which was elevated to 29.5 +/- 1.4 degrees C in SPM treated with cholesterol glucoside (50 microM) and abolished in SPM treated with cholesterol (5 microM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F- and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 microM) produced an increased packing of the bulk lipids, while cholesterol (5 microM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.