Induction of t-PA Synthesis with intravenous bolus injection of vitamin A palmitate in vitamin a deficient rats

Retinoic acid and vitamin A palmitate induce tissue-type plasminogen activator (t-PA) synthesis in cultured human umbilical vein endothelial cells (HUVEC) in vitro without alteration of plasminogen activator inhibitor-] (PAI-1) synthesis. Therefore the effect of intravenous bolus injection of water-miscible vitamin A palmitate on the blood fibrinolytic system was investigated in normal and vitamin A deficient rats. In 5 normal rats, injection of 200000 IU/kg of vitamin A palmitate did not induce a significant increase in euglobulin fibrinolytic activity, t-PA antigen and PAI activity in plasma nor of t-PA and PAI-1 mRNA in the heart within 240 min after injection. In groups of 3–6 vitamin A deficient rats, injection of 200000IU/kg of vitamin A palmitate induced a significant increase in t-PA antigen in plasma (1.9-fold after 60 min and 2.9-fold after 120 min), but no significant increase in plasma euglobulin fibrinolytic activity. A 2-fold increase in PAI activity was observed 90 min after injection of both vitamin A palmitate as well as of its solvent, suggesting that this induction was non-specific. The increase in t-PA antigen coincided with a significant increase in t-PA mRNA in heart tissue: 2.3-fold after 60 min and 4.3-fold after 120 min. PAI-1 mRNA in heart tissue increased 3.6-fold 90 min after injection with vitamin A palmitate but, surprisingly, not after injection of solvent.It is concluded that intravenous bolus injection of vitamin A palmitate induces a specific increase of t-PA mRNA in the heart and of t-PA antigen secretion in plasma of vitamin A deficient rats.     

Tritiation of protein antigens of Plasmodium knowlesi schizont-infected erythrocytes using pyridoxal phosphate-sodium boro[3H]hydride

The protein antigens of Plasmodium knowlesi schizont-infected red blood cells (SI-RBCs) and normal RBCs were compared using the pyridoxal phosphate/NaB³H₄ method. Permeation of the outer SI-RBC membrane by the pyridoxal phosphate anion was enhanced since unlike normal RBCs it was not possible to exclusively label surface membrane proteins without concurrent haemoglobin labelling. Under conditions of minimal haemoglobin labelling a subset of total susceptible SI-RBC proteins (M_r 125 000, 50 000, 45 000 and 30 000) were labelled that were absent from normal RBCs and which may be surface proteins. The M_r 125 000 band labels much more readily than Band 3, the normal anion transporter, suggesting that it may be a new anion transporter in the SI-RBC membrane. At higher pyridoxal phosphate concentrations additional bands (M_r 230 000, 180 000, 165 000, 145 000, 107 000 and 72 000) were labelled exclusively with SI-RBCs. The new pyridoxal phosphate-labelled proteins had altered electrophoretic mobility and reduced Coomassie Blue staining, both properties assisting in their identification. Antigen analysis using Protein A-Sepharose and sera from infected monkeys demonstrated that all new ³H-labelled proteins were SI-RBC-specific antigens. One very high M_r antigen (> 250 000) recognized only by homologous strain antisera may represent a strain-specific antigen.     

The fibrinolytic response to exercise at diagnosis and after 12 months in patients with type 2 diabetes mellitus

Objective: To determine fibrinolytic activity and exercise stimulated fibrinolytic capacity in patients with type 2 diabetes mellitus at diagnosis and after 12 months treatment aimed at improving glycaemic control.Subjects: Thirteen patients referred to the hospital diabetic out-patient department with a new diagnosis of type 2 diabetes mellitus were selected for study.Methods: Basal fibrinolytic activity and exercise stimulated fibrinolytic capacity were measured at diagnosis and after 12 months of interventional therapy.Results: Compared with controls basal fibrinolytic activity was depressed in the diabetic patients due to increased levels of PAI-1:Ag 15.1(4.6–20) versus 8.4 (3.0–9.9) ng/ml, p<0.05 and PAI,17.6 (10.9–26.8) versus 6.6 (4.8–13.6) IU/ml, p<0.01. PAI was related to fasting plasma insulin levels r=0.8, p<0.001 and body mass index r=0.7, p<0.01 at diagnosis, but not triglycerides or blood pressure. Median t-PA:Ag was also elevated in the diabetic group 9.8 (6.3–12.1) versus 4.5 (3.1–7.1) ng/ml, p<0.001. The percentage change in ECLT with exercise was inversely proportional to the degree of insulin resistance r=−0.08, p<0.001 and fasting plasma insulin r=−0.65, p--0.02, at diagnosis. Despite an improvement in glycosylated haemoglobin, 8.7 (1.8) to 7.0 (1.2) %, p=0.008, over 12 months, the ECLT increased from 290 (220–315) to 360 (316–375) mins, p<0.05. This was associated with an increase in the PAI-1:Ag/t-PA:Ag ratio from 1.92 (1.63) to 2.48 (0.97), p=0.025. Although t-PA release due to exercise was reduced in the diabetic group (38%) compared to non-diabetic controls (82%) p<0.001, similar results were found after 12 months 54.5% to those at diagnosis 38%, (NS).Conclusion: This study has shown that in a selective group of type 2 diabetic patients, exercise stimulated fibrinolytic capacity is maintained over a 12 month period despite a deterioration in basal fibrinolytic activity.     

Partial chemical characterization of the carbohydrate moieties in Leishmania adleri glycoconjugates

Promastigotes of Leishmania adleri were submitted to an extraction procedure providing different carbohydrate-containing extracts. The purified aqueous extract showed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a complex peptide pattern but carbohydrate was present only in bands of M_r ≈ 45 000–50 000 and 13 500. Methylated derivatives of the hexose components in this extract, analysed by mass spectrometry, suggest the presence of short sugar chains of α-d-mannopyranose and a branched α-d-mannan. The phenol extract, released in the aqueous layer a chloroform/methanol/water soluble complex contained 25% protein, 17% phosphate, 11% glucosamine, uronic acid and 61% neutral carbohydrate, and a chloroform/methanol/water insoluble fraction consisting of a glycoprotein M_r ≈ 22 000 and a proteic doublet M_r ≈ 58 000–66 000. A polysaccharide, showing galactose as predominant sugar, was released through alkaline extraction corresponding to a branched, mainly 1→3 linked galactan associated with α-d-mannopyranosyl units.     

Radioiodination of new protein antigens on the surface of Plasmodium knowlesi schizont-infected erythrocytes

Schizont-infected red blood cells (SI-RBCs) from Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta) have been radioiodinated and the ¹²⁵I-proteins and ¹²⁵I-antigens compared with those of uninfected RBCs. New ¹²⁵I-proteins of M_r 230 000, 180 000, 165 000, 155 000, 135 000, 107 000, 72 000 and 65 000 were shown to be parasite-dependent components of SI-RBCs, the M_r 230 000 and 72 000 bands being quantitatively the major new components. New ¹²⁵I-antigens of SI-RBCs that were absent from uninfected cells and were only immunoprecipitated by sera from infected monkeys had M_r 230 000, 200 000, 180 000, 165 000, 155 000, 135 000, 130 000, 107 000, 72 000, 65 000 and 47 000. The following approaches were used to test which new radioiodinated components are on the SI-RBC outer membrane: analysis of radioactivity in haemoglobin; electron microscopic analysis of the integrity and purity of the SI-RBCs; treatment of intact ¹²⁵I-SI-RBCs with trypsin to ascertain the sensitivity of new proteins to exogenous protease; immunoprecipitation of antigen-antibody complexes after addition of antibody to intact ¹²⁵I-SI-RBCs. Although each test has inherent disadvantages, the accumulated evidence suggests that many, if not all of the new antigens identified for the first time in this report are on the SI-RBC outer membrane. The SICA variant antigen on P. knowlesi SI-RBCs was not identified by this approach.     

The occurrence and synthesis of octopamine and catecholamines in the cestode Hymenolepis diminuta

We have defined the surface protein antigens on Plasmodium gallinaceum zygotes using radioiodination methods and rabbit anti-zygote serum which blocks transmission of the parasite to Aedes aegypti mosquitoes. Fifteen protein bands (1–15) in the molecular weight range of 40 000–240 000 and one band at the bromophenol blue dye marker were labelled by the lactoperoxidase and IODOGEN (1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril) methods. The localization of these radioiodinated components on the cell surface was confirmed in two ways: (1) They were completely degraded by trypsin or Streptomyces griseus protease treatment of intact viable zygotes. (2) They were immunoprecipitated following incubation of intact zygotes with antibody prior to detergent solubilization. Reactivity with immune rabbit serum demonstrated that the major surface immunogens were components with M_r 240 000, 200 000, 180 000, 80 000, 55 000, 50 000 and the band at the dye marker. A band of M_r 180 000 was shown immunologically to be a host serum protein selectively adsorbed to the zygote surface. The identity of this protein and the significance of its adsorption to the surface of the zygote are unknown.     

Toward Standardization of BK Virus Monitoring: Evaluation of the BK Virus R-gene Kit for Quantification of BK Viral Load in Urine,Whole-Blood,and Plasma Specimens

Screening of BK virus (BKV) replication is recommended to identify patients at increased risk of BKV-associated diseases. However, the heterogeneity of molecular techniques hinders the establishment of universal guidelines for BKV monitoring. Here we aimed to compare the performance of the CE-marked BK virus R-gene kit (R-gene) to the performance of our in-house assay for quantification of BKV DNA loads (BKVL). A 12-specimen panel from the Quality Control for Molecular Diagnostics (QCMD) organization, 163 urine samples, and 88 paired specimens of plasma and whole blood (WB) from transplant recipients were tested. Both the R-gene and in-house assays showed a good correlation within the QCMD panel (r = 0.995 and r = 0.989, respectively). BKVL were highly correlated between assays, although positive biases were observed with the in-house assay in analysis of urine (0.72 ± 0.83 log₁₀ copies/ml), plasma (1.17 ± 0.63 log₁₀ copies/ml), and WB (1.28 ± 0.37 log₁₀ copies/ml). Recalibration with a common calibrator significantly reduced the bias in comparisons between assays. In contrast, BKVL was underestimated with the in-house PCR in eight samples containing BKV genotype II, presenting point mutations at primer-annealing sites. Using the R-gene assay, plasma and WB specimens were found to be equally suitable for quantification of BKVL, as indicated by the high correlation coefficient (r = 0.965, P < 0.0001). In conclusion, the R-gene assay demonstrated reliable performance and higher accuracy than the in-house assay for quantification of BKVL in urine and blood specimens. Screening of BKV replication by a well-validated commercial kit may enable clinical laboratories to assess viral loads with greater reproducibility and precision.     

Phospholipase C-mediated inhibition of the M-potassium current by muscarinic-receptor activation in the vestibular primary-afferent neurons of the rat

The activation of the efferent vestibular system modifies the basal discharge and the dynamic response of primary-afferent neurons to head motion and gravitational stimuli. The efferent input to afferent neurons is mediated primarily by cholinergic synapses that activate both muscarinic and nicotinic receptors. Previously we had shown that the muscarinic-acetylcholine-receptor (mAChR) activation modulates the low-voltage-activated M-type potassium current (I_K,M) in the vestibular-afferent neurons. In this work we studied the second-messenger system mediating the inhibition of I_K,M after mAChR activation. For this, voltage and current-clamp recordings were obtained in the cultured vestibular-afferent neurons of the rat. The I_K,M was measured during its deactivation. Response to current-pulse injection was also studied. The use of the mAChR agonist oxotremorine-M significantly reduced the amplitude of the I_K,M and modified the discharge response to current pulses from single spike to multiple spiking, reducing the adaptation of the electrical discharge. The intracellular perfusion of the phospholipase C (PLC) inhibitor U73122 significantly attenuated the inhibitory action of the mAChR receptor agonist oxotremorine-M. Its inactive analog U73343 produced no significant action. The use of the phosphatidylinositol 4,5 bis-phosphate (PIP₂) scavenger poly-l-lysine also led to a significant reduction of the I_K,M. Our results show that the mAChR mediated activation of PLC and subsequent PIP₂ depletion (caused by its hydrolysis), modulates the I_K,M in the vestibular-afferent neurons, modifying their discharge response dynamics to current-pulse injection.     

The oxygen consumption of mammalian non-myelinated nerve fibres at rest and during activity

1. A study has been made of the oxygen consumption of non-myelinated nerve fibres of rabbit desheathed cervical vagus nerves at rest and during activity.2. The average resting oxygen consumption (Q_r) was 0·0924 μmole/g. min at 21° C. Stimulation for 1-3 min at 3/sec caused an extra oxygen consumption (Q_s) of 816 p-mole/g.shock.3. When the frequency of stimulation was increased, to 10/sec and 30/sec, Q_s fell. When the frequency was decreased, to 1/sec and 0·3/sec, Q_s increased slightly.4. When the temperature was decreased, Q_r fell; when the temperature was increased, Q_s also increased. Temperature similarly affected Q_s with high frequencies of stimulation, but had relatively little effect on Q_s at low frequencies of stimulation.5. An isolated single shock seemed to produce an increase in oxygen consumption of about 1200 p-mole/g, and this value was largely independent of temperature.6. When part of the sodium in the Locke solution was replaced by barium, Q_r decreased (by 12%) whereas Q_s increased (by 87%).7. Veratrine (1 μg/ml.) increased both Q_r (by 142%) and Q_s (by 361%).8. Acetylcholine (1·7 mM) increased Q_r (by 32%).9. When nerves were transferred to potassium-free solutions there was little change in Q_r, and Q_s fell slightly (by 8%).10. When the potassium concentration in the Locke solution was increased 4-fold, Q_r increased (by 27%).11. Salicylate (1-10 mM) increased Q_r (by 24%) and abolished Q_s.12. When the sodium of Locke solution was replaced by lithium, Q_r decreased (by 19%) and Q_s was abolished.13. In sodium-Locke solution ouabain (100 μM) decreased Q_r (by 26%) and abolished Q_s. In lithium-Locke solution ouabain also decreased Q_r (by 28%).14. All or nearly all of the oxygen consumed at rest or during activity seemed to be used to pump potassium ions into, and sodium ions out of, the axoplasm.15. The K/O₂ ratio during pumping was about 5·0.     

A major change in surface antigens during the maturation of newborn larvae of Trichinella spiralis

Newborn larvae of Trichinella spiralis were collected for 30 min from female worms in culture, incubated in vitro for various times up to 18 h, and surface-labelled with iodine. The detergent-solubilised products were examined by SDS-polyacrylamide gel electrophoresis. At time periods up to 6 h these larvae expressed only one M_r 64 000 iodine-labelled surface protein. Some time between 6 h and 18 h a further three components (apparent M_r 58 000, 34 000 and 32 000) became accessible to surface labelling. All four of these components are antigenic in that they can be immunoprecipitated with T. spiralis immune sera. Tryptic peptide analysis revealed that the 32 and 34 kDa antigens were structurally very similar, but the 58 and 64 kDa proteins differed from each other and the 32–34 kDa pair. Thus T. spiralis not only undergoes a total change in surface antigens between moults, but also major changes in surface antigen expression within one stage.     

The dependence on external cations of the oxygen consumption of mammalian non-myelinated fibres at rest and during activity

1. A study has been made of the effect of potassium and other cations on the oxygen consumption of rabbit desheathed vagus nerves at rest and during activity.2. In normal Locke solution (containing 5·6 mM-K) the resting oxygen consumption, Q_r, was 0·0903 μmole/g wet wt. min. The extra oxygen consumed as a result of stimulation, Q_s, at 3 stimuli/sec was about 600 p-mole/g.impulse; at 30 stimuli/sec it was less, being 240 p-mole/g.impulse.3. Only a fraction of Q_r (18% at 5·6 mM-K) was sensitive to ouabain (1 mM). The ouabain-sensitive component, however, increased as the external potassium concentration was increased, in the range 0-100 mM. Q_s was virtually abolished by ouabain.4. Reduction of the external potassium concentration from 5·6 mM to zero reduced Q_r (by 10%) and increased Q_s, but the changes were scarcely significant statistically.5. The conclusions were drawn: that Q_s reflected the pumping of cations to restore the ionic imbalance following activity, particularly reflecting the extrusion of sodium ions from the fibre; that this pumping was normally absolutely dependent on the presence of potassium externally and that no pumping could occur in its absence; and that Q_s was not reduced to zero in ostensibly potassium-free solutions because enough potassium was released into the periaxonal space during activity to maintain pumping.6. Thallium, rubidium, caesium and lithium could replace potassium and allowed pumping to occur.     

Immunohistochemical angiogenic biomarkers in hepatocellular carcinoma and cirrhosis: correlation with pathological features

OBJECTIVE:To investigate immunohistochemical markers of angiogenesis and their association with pathological prognostic features in hepatocellular carcinoma and cirrhotic liver.METHODS:Vascular endothelial growth factor, CD105, and cyclooxygenase-2 were immunohistochemically detected in 52 hepatocellular carcinoma tissue samples and 48 cirrhotic liver tissue samples. Semiquantitative measurements of vascular endothelial growth factor and cyclooxygenase-2 were evaluated considering the degree and intensity of immunostaining based on a 7-point final scoring scale. CD105 microvascular density (MVD-CD105) was measured using automated analysis. Morphological aspects evaluated in the hepatocellular carcinoma samples included size (≤2 and >2 cm), differentiation grade, and microvascular invasion.RESULTS:The mean vascular endothelial growth factor immunoreactivity score was slightly higher in the hepatocellular carcinoma samples (4.83±1.35) than the cirrhotic liver (4.38±1.28) samples. There was a significant and direct correlation between these mean scores (r_s=0.645, p=0.0001). Cyclooxygenase-2 was expressed in all the cirrhotic liver samples but was only found in 78% of the hepatocellular carcinoma samples. The mean cyclooxygenase-2 score was higher in the cirrhotic liver samples (4.85±1.38) than the hepatocellular carcinoma samples (2.58±1.68), but there was no correlation between the scores (r_s=0.177, p=0.23). The mean CD105 percentage in the hepatocellular carcinoma samples (11.2%) was lower than that in the cirrhotic samples (16.9%). There was an inverse relationship in MVD-CD105 expression between the hepatocellular carcinoma and cirrhotic samples (r_s=-0.78, p=0.67). There were no significant associations between vascular endothelial growth factor expression and morphological characteristics. Cyclooxygenase-2 and CD105 were associated with hepatocellular carcinoma differentiation grade (p=0.003 and p=0.05, respectively).CONCLUSION:Vascular endothelial growth factor, cyclooxygenase-2, and MVD-CD105 were highly expressed in cirrhotic liver compared to hepatocellular carcinoma and might be involved in liver carcinogenesis. Additionally, cyclooxygenase-2 and CD105 might be involved in hepatocellular carcinoma differentiation grade.     

Disturbances of Haemostasis in Diabetes Mellitus

Diabetes mellitus is associated with disturbances in haemostasis that could contribute to the development of thrombotic complications.The present study was undertaken to determine the behavior of coagulation variables and fibrinolytic system in diabetes mellitus. Forty five diabetic patients and forty five matched controls were evaluated by doing the following haemostatic parameter, prothrombin time, partial thromboplastin time, thrombin time, coagulation factors assay II, VII, IX, & plasma fibrinogen, ADP-induced platelet aggregation, protein C, a₂- antiplasmin, PAI and FDPs. Generally diabetic patients have high levels of fibrinogen, a₂- antiplasmin, & PAI and lower level of protein C. Other haemostatic parameters did not show statistically significant difference between diabetic patients and control group. Significantally elevated levels of PAI, a₂- antiplasmin together with low protein C level in diabetic patients may result in the disturbance of haemostatic balance favoring thrombotic events. Conclusion: High levels of plasma fibrinogen, a₂A- antiplasmin with low plasma protein C activity could lead to a prothrombotic tendency in insulin dependent diabetic patients. Moreover, in non-insulin dependent diabetic patients, the above mentioned parameters together with high levels of ADP-induced platelet aggregation and plasminogen activator inhibitor may increase the risk of thrombotic complications. Obesity can be considered as an additional risk factor for development of thrombosis in diabetic patients.     

Decreased Proinflammatory Cytokines Production in Children with Complicated Parapneumonic Pleural Effusion after Intrapleural Fibrinolytic Treatment

Intrapleural fibrinolytic therapy (IFT) provides clinical benefit in the treatment of complicated pleural parapneumonic effusion (CPE). Whether IFT influences the proinflammatory cytokines production and fibrinlytic activity is currently unclear. Therefore, we collected pleural effusion samples from CPE patients with IFT (study group) and patients without IFT (control group). A membrane human inflammatory cytokines array kit was used to compare the difference of targeted cytokine production between these two groups. Enzyme-linked immunosorbent assay (ELISA) methods were used for quantitative analysis of targeted cytokines and fibrinolytic enzymes. The results showed there were no significant differences between the study (n = 16) and control (n = 14) groups in patients’ demographic data. After fibrinolytic therapy, the patients in the study group had significant lower plasminogen activator inhibitor (PAI) level (732.36?±?254.09 ng/mL vs 1,509.36?±?1,340.11 ng/mL, p?<?0.05) and higher urokinase plasminogen activator (u-PA) level (75.56?±?41.70 ng/mL vs 6.87?±?5.07 ng/mL, p?<?0.05) than they did before treatment. Moreover, the tissue inhibitors of metalloproteinase-2 (TIMP-2) (1,560.03?±?403.49 pg/mL vs 3,686.45?±?1,263.83 pg/mL, p?<?0.05) and inflammatory chemokine, regulated on activation normal T-cell expressed and secreted/chemokine (C-C motif) ligand 5 (RANTES), (293.58?±?212.93 pg/mL vs 749.27?±?53.79 pg/mL, p?<?0.05), were also significantly lower in the study group after fibrinolytic therapy, but not in the control group. In conclusion, intrapleural fibrinolytic treatment with urokinase could enhance fibrinolytic activity and decrease TIMP-2 and RANTES production.     

Asthma is associated with reduced fibrinolytic activity,abnormal clot architecture,and decreased clot retraction rate

The aim of this study was to assess whether steroid‐naïve asthma modulates hemostasis. We evaluated the clot retraction rate (CRR), fibrinolysis rate (FR), clot density (CD) (by confocal microscopy), plasma levels of plasminogen activator inhibitor (PAI‐1), and factor XIII (FXIII), NO in exhaled breath (FE_NO), spirometry (FEV₁) and eosinophil count (EOS) in 36 patients with allergic, steroid‐naïve asthma and in 34 healthy controls. We observed significantly (P < 0.001) reduced CRR, FR, and FEV₁ and increased FE_NO, EOS, PAI‐1, FXIII, and CD in patients with asthma compared with controls. In patients with asthma, FR negatively correlated with CD, FXIII, PAI‐1, FE_NO, and EOS and positively with FEV₁. FXIII positively correlated with CD. Clot retraction rate negatively correlated with FE_NO and positively with FEV₁ (all P < 0.001). These novel findings suggest that asthma itself is associated with decreased CRR and reduced fibrinolytic potential resulting from alterations in clot architecture and elevated levels of plasma FXIII and PAI‐1.     

Untersuchung zur Frage von persistierenden Veränderungen der Fibrinolyseparameter t-PA und PAI bei Patienten nach juvenilem ischämischen cerebralem Insult

Summary In diseases associated with thrombotic or thromboembolic complications, a reduction in the fibrinolytic potential may contribute to the risk to develop thrombosis.To investigate whether iuvenile cerebral infarction is associated with a permanent defect of the fibrinolytic system we measured the main components of the fibrinolytic system, tissue plasminogen activator (t-PA) and its fast acting inhibitor (PAI) in plasma samples of 21 patients (aged 21–44 years) 3–24 months after the acute event. The data obtained were compared to those from thirteen healthy young volunteers (22–46 years). A direct effect of known risk factors on the fibrinolytic system could be excluded because patients avoided their risk factors immediately after the ischemic cerebral attack. Hypertension and the combination of oral contraceptives and smoking had been the most striking original risk factors.Levels of t-PA antigen and t-PA activity before and after venous occlusion, or PAI activity were not different between patients and controls suggesting that at least a permanent decrease in the activity of the fibrinolytic system does not exist in these patients. However, our findings do not exclude that a temporary defect in fibrinolysis might have contributed to the acute onset of the thrombotic cerebral event possibly induced by the risk factors originally present.

---

Abkürzungen t-PA tissue plasminogen activator - PAI plasminogen activator inhibitor - RIND reversibles ischämisches neurologisches Defizit - KS kompletter Schlaganfall - TIA transitorisch ischämische Attacke     

Determination of α2-antiplasmin-plasmin complex in human plasma with an enzyme-linked immunosorbent assay

A novel enzyme-linked immunosorbent assay (ELISA) for quantification of α₂-antiplasmin-plasmin complex (APP) in undiluted plasma was developed. The assay follows the sandwich principle and uses two different antibodies directed against plasmin-modified α₂-antiplasmin and plasminogen, respectively. The antibodies bind selectively to the corresponding antigen moieties of APP. The assay was calibrated with definite concentrations of preformed purified APP added to APP-poor plasma. The lower limit of sensitivity of the assay was 10ng/ml. Mean coefficients of variation of 5.8% (intraassay) and 7.2% (interassay) were found for APP concentrations between 50 and 5000 ng/ml. A reference range from 80–470 ng/ml was calculated from APP concentration in plasma samples from 178 healthy donors (mean value±SD: 210±88). In plasma samples from patients during thrombolytic therapy, APP was found up to 20000 ng/ml. From our data we conclude that quantification of APP can be a sensitive tool for specific detection of an activation of the fibrinolytic system.     

Tumour necrosis factor α production by rat blood and itsex vivo pharmacological modulation

Rat blood was investigated as a suitable test system for the discovery of inhibitors of tumour necrosis factor α (TNFα) biosynthesis. Lipopolysaccharide (LPS) caused a concentration- and time-dependent stimulation of TNFα production by heparinised rat blood with peak levels (1000–5000 U/ml; L929 bioassay) at 6h. Bioactive material was neutralised with a polyclonal rabbit anti-murine TNFα antibody which cross-reacts with rat TNFα. Dexamethasone, pentoxifylline and denbufylline inhibited TNFα production with IC₅₀s of 6.0±2.0 nM, 20.6±8.00 μM and 138.0 nM, respectively. When rats were dosed p.o. with dexamethasone or pentoxifylline or i.p. with denbufylline and 1.5h later TNFα production was assessedex vivo by LPS-stimulated blood, a dose-related inhibition of TNFα production occurred with ID₅₀s of approximately 0.08, 250.0 and 5.0 mg/kg, respectively. These results demonstrate that rat blood provides a useful test system for the detection andex vivo evaluation of inhibitors of TNFα biosynthesis.