Targeted therapy of cognitive deficits in fragile X syndrome.

Breaking an impasse in finding mechanism-based therapies of neuropsychiatric disorders requires a strategic shift towards alleviating individual symptoms. Here we present a symptom and circuit-specific approach to rescue deficits of reward learning in Fmr1 knockout mice, a model of Fragile X syndrome (FXS), the most common monogenetic cause of inherited mental disability and autism. We use high-throughput, ecologically-relevant automated tests of cognition and social behavior to assess effectiveness of the circuit-targeted injections of designer nanoparticles, loaded with TIMP metalloproteinase inhibitor 1 protein (TIMP-1). Further, to investigate the impact of our therapeutic strategy on neuronal plasticity we perform long-term potentiation recordings and high-resolution electron microscopy. We show that central amygdala-targeted delivery of TIMP-1 designer nanoparticles reverses impaired cognition in Fmr1 knockouts, while having no impact on deficits of social behavior, hence corroborating symptom-specificity of the proposed approach. Moreover, we elucidate the neural correlates of the highly specific behavioral rescue by showing that the applied therapeutic intervention restores functional synaptic plasticity and ultrastructure of neurons in the central amygdala. Thus, we present a targeted, symptom-specific and mechanism-based strategy to remedy cognitive deficits in Fragile X syndrome.

Evaluation of hyperprolactinaemia with the use of the intervals for prolactin after macroforms separation.

Macroprolactin (MaPRL) - a complex of monomeric prolactin (PRL) with immunoglobulin G, may be a cause of laboratory diagnosed hyperprolactinaemia. To quantify MaPRL, a precipitation with polyethylene glycol may be performed. This method involves calculating of recovery ratio but the cut-off value is not precisely determined. Moreover, it is proposed that the assessment of macroprolactinaemia should include also the evaluation of real PRL concentration which means the level of the hormone after macroforms separation. The study included 245 patients with hyperprolactinaemia, in whom precipitation was performed. A recovery ratio ≤40% indicated macroprolactinaemia. The real PRL concentrations of the studied subjects were compared with reference ranges suggested by the assay manufacturer and with new intervals for PRL after macroforms separation. On the base of the recovery ratio after the precipitation, macroprolactinaemia was detected in 21 persons. In these patients true hyperprolactinaemia (elevation of real PRL concentration above manufacturer's reference ranges) was noted in 9 cases. Among 224 patients with a recovery >40%, real PRL concentration turned out to be within the manufacturer's reference range (pseudohyperprolactinaemia) in 36 persons. The new intervals for PRL after macroforms separation were about 20% lower than the manufacturer's reference ranges. After applying new ranges in patients with macroprolactinaemia, true hyperprolactinaemia was observed in 14 persons, while in the group without MaPRL dominance, pseudohyperprolactinaemia was noted in 5 patients. The use of the recovery ratio only to recognize macroprolactinaemia may lead in some subjects to the misclassification of the results. For that reason the assessment of the PRL concentration after macroforms separation that can help to distinguish true hyperprolactinaemia and pseudohyperprolactinaemia, seems to be reasonable. To evaluate the real PRL concentration, the reference intervals suggested by the manufacturer of immunoassay might be used. However, possibly better means to diagnose patients with hyperprolactinaemia accurately is using an appropriate range for the concentration of PRL after macroforms separation.

Ghrelin but not obestatin regulates insulin secretion from INS1 beta cell line via UCP2-dependent mechanism.

The mitochondrial UCP2 mediates glucose-stimulated insulin secretion by decreasing intracellular ATP/ADP ratio. Insulin secretion is a tightly regulated process. Ghrelin, as well as obestatin, were intensively studied to determine their ability to modify insulin secretion. Ghrelin is considered to be an inhibitor of insulin release from pancreatic islets, however little is known about the effects of obestatin. In our study we demonstrate the stimulating effects of both peptides on insulin secretion in INS1 cells. Furthermore, we investigate the potential role of UCP2 in mediating the effects of both peptides on insulin secretion. UCP2 mRNA expression was down-regulated by ghrelin in the presence of 26.4 mM glucose, however it was unchanged after obestatin treatment. Our results confirm that UCP2 could be involved in the stimulating effect of ghrelin on insulin release from INS1 cells.

Increased coagulation activity of the pleura after tube drainage and quinacrine instillation in malignant pleural effusion.

Chronic malignant pleural effusions are usually treated with an intrapleurally administered irritant that creates an inflammatory reaction. The induced inflammation results in fibrin deposition and termination of fluid exudation. In the present study several factors in the coagulation system in the pleural fluid were followed during treatment with tube drainage and quinacrine instillation into the pleural space. In the chronic exudative phase before treatment, both thrombin activity and fibrinopeptide A (FPA), were present at low levels. During treatment the levels increased markedly. Beta-thromboglobulin, a platelet marker, showed a similar pattern. Prothrombin, antithrombin III, prekallikrein and kallikrein inhibiting activity showed no such variations in activity. The high thrombin activity and FPA level induced by treatment reflect an active process of fibrin formation which seems to play an important role in arresting chronic pleural exudation.

The acute effect of insulin on tissue plasminogen activator and plasminogen activator inhibitor in man.

The present study was performed to elucidate the acute effect of insulin on levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor of endothelial cell type (PAI-1). Nine middle-aged, non-obese and non-smoking men were studied during a hyperinsulinemic, euglycemic glucose clamp for 2 h. Plasma insulin level during the clamp averaged 84 +/- 12 mU/l and euglycemia was maintained at 4.9 +/- 0.6 mmol/l. The t-PA activity gradually increased (75% mean increase after 2 h, p less than 0.001) and the PAI-1 activity decreased (49% mean decrease after 2 h, p less than 0.001) during the clamp. t-PA activity decreased and PAI-1 activity increased after the insulin infusion was ceased, but they were still 48% higher and 38% lower, respectively, after 60 min. PAI-1 and t-PA activities were not affected by saline infusion for 2 h. Thus, acute changes in the insulin levels lead to rapid alterations in the fibrinolytic system even when euglycemia is maintained. These effects may be induced by insulin itself or by the concomitant activation of the sympatho-adrenal system during the euglycemic clamp.

Pleural fibrinolytic activity is decreased in inflammation as demonstrated in quinacrine pleurodesis treatment of malignant pleural effusion.

Chronic malignant pleural exudation is generally characterized by little or no fibrin deposition. However, during induced inflammation, fibrin deposition concomitant with cessation of exudation is seen. To judge the involvement of the fibrinolytic system in this process, concentrations of fibrinolytic factors were followed in 10 patients during treatment by quinacrine instillation and tube drainage. Plasminogen and alpha-2-antiplasmin were found in low concentrations and did not show significant changes during treatment. The plasminogen activator inhibitor PAI-1, which plays an important role in the regulation of fibrinolysis, was also studied during treatment. Before treatment the concentration of PAI-1 was 21.7 +/- 12.0 (mean +/- SD) AU/ml and it increased to 86.9 +/- 25.9 AU/ml 6 h after quinacrine instillation. D-dimer, a product of the lysis of fibrin, was found in high concentrations before treatment (62.7 +/- 25.5 micrograms/ml) and in low concentrations 6 h after treatment (12.2 +/- 7.9 micrograms/ml). Thus, it was possible to demonstrate that the fibrinolytic system is activated during chronic malignant pleural exudation and, furthermore, that the activity decreases during induced inflammation.

Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for 'second-site' interactions.

The reactions between plasminogen-activator inhibitor (PAI) and different plasminogen activators were studied in the presence of chromogenic peptide substrates for the enzymes. Our findings suggest that the rate constants for the reactions of PAI with single-chain tissue plasminogen activator (tPA), two-chain tPA, high-Mr urokinase and low-Mr urokinase are high and quite similar (1.6 X 10(7)-3.9 X 10(7) M-1.s-1). A free active site in the enzymes seems to be necessary for their reaction with PAI. Amino acids with antifibrinolytic properties did not interfere with the reactions. However, di-isopropyl phosphorofluoridate-inactivated tPA inhibited the reaction between PAI and all plasminogen activators in a similar way. These findings clearly demonstrated that a 'second-site' interaction, in addition to that between the enzyme active site and the inhibitor 'bait' peptide bond, is of importance for the high reaction rate. The reaction rate between PAI and single-chain tPA in the presence of an activator substrate (D-Ile-Pro-Arg p-nitroanilide) was decreased in the presence of fibrin. Fibrin caused a decrease in the Km for the single-chain tPA-substrate reaction. As a consequence, the 'free' concentration of single-chain tPA in the system decreased in the presence of fibrin, affecting the reaction rate between PAI and single-chain tPA. The phenomenon might be of physiological relevance, in the sense that single-chain tPA bound to fibrin in the presence of plasminogen would be protected against inactivation by PAI.

Immunological relationship between plasminogen activator inhibitors from different sources.

Polyclonal antibodies have been raised against the inhibitor moiety in the purified complex between tissue plasminogen activator and its fast inhibitor (PA-inhibitor) in human plasma/serum. A radioimmunoassay for quantitation of PA-inhibitor antigen was developed. The polyclonal antiserum and a previously described monoclonal antibody against the PA-inhibitor (14) have been used to study the immunological relationship between PA-inhibitors from plasma, serum, platelets, placenta extract and conditioned media from Hep G2 and HT 1080 cells. It was demonstrated that the ratio between PA-inhibitor activity and antigen varied considerably between the different sources. In the plasma samples studied, similar activity and antigen concentrations were found, suggesting that the PA-inhibitor in these samples mainly was in an active form. On the other hand the other sources seemed to contain variable amounts of inactive PA-inhibitor forms. Immunoadsorption experiments revealed that the PA-inhibitor (activity and antigen) from all the sources were specifically bound to the insolubilized antibodies (polyclonal and monoclonal). In no case, however, could active PA-inhibitor be eluted from the immunoadsorption columns. Also the competitive radioimmunoassays suggested that the PA-inhibitors from the different sources studied, were closely immunologically related.

Monoclonal antibodies towards the fast inhibitor of tissue plasminogen activator from human plasma and serum.

Hybridoma cells were produced by fusing mouse myeloma cells (SP 2/0-Ag 14) with spleen cells from a Balb/c mouse, previously immunized with the partially purified complex between tissue plasminogen activator (t-PA) and its fast inhibitor from human plasma (serum). Screening with a radioimmunoassay revealed a number of hybridomas secreting antibodies directed towards the complex. Of these, about 1/3 reacted both with the complex and t-PA, whereas about 2/3 reacted only with the complex. Three of the latter hybridomas, producing antibodies directed towards the inhibitor-moiety in the complex have been cloned and the antibodies were studied in detail. PA-inhibitor activity in plasma or serum and t-PA/PA-inhibitor complex could be specifically adsorbed on all three insolubilized monoclonal antibodies (MC1, MC2 and MC3). None of the antibodies seems to be directed against structures of vital importance for the functional activity of the PA-inhibitor. In accordance with this finding the antibody with the highest avidity (MC1) reacts equally well with the PA-inhibitor alone or in complex with t-PA. A radioimmunoassay was developed with this antibody and significant displacement was obtained with samples with PA-inhibitor concentrations above 2 AU/mL. In 13 plasma samples with different levels of PA-inhibitory activity a significant correlation was obtained when comparing this activity with the PA-inhibitor antigen as measured with the radioimmunoassay (r = 0.88).

Determination of tissue plasminogen activator and its "fast" inhibitor in plasma.

We describe efficient, accurate methods for specific determination of tissue plasminogen activator (t-PA, EC 3.4.21.31) and its "fast" inhibitor in plasma. In this coupled assay, a sample containing t-PA is incubated with plasminogen, a plasmin (EC 3.4.21.7) substrate of low Km and high Kcat, and fibrin as a stimulator. The inhibitor of t-PA is determined by incubating the sample with a known amount of t-PA in excess, then determining the residual t-PA. Both t-PA and t-PA inhibitor can be determined in many samples simultaneously within a few hours. These assays are modifications of procedures described by us (Clin Chim Acta 1983;127:279-88 and Thromb Res 1983;31:427-36). Their accuracy as assessed by analytical recovery of pure t-PA added to blood samples (91 +/- 4%) or of partly purified inhibitor added to plasma samples (102 +/- 10%) is satisfactory, as is their precision. For the t-PA assay the CV was 1.6% (within run) or 4.1% (between run). The corresponding values for the inhibitor assay were 4.5% (within run) or 8.4% (between run) if the inhibitor concentration exceeded 3 arb. units/mL.

Studies on blood coagulation activity during storage of red cell products intended for transfusion.

Preservation of red blood cells in special media, such as saline-adenine-glucose-mannitol (SAGM) solution involves storage at a citrate and plasma protein concentration much lower than found in normal CPD or CPD-adenine plasma. In the present study, the effects on the coagulation and other connected enzymatic systems in SAGM medium have been investigated. Significantly higher levels of fibrinopeptide A (FPA) and plasma kallikrein activity were observed in the SAGM medium compared to CPD plasma, indicating an increased enzymatic activation in the SAGM medium. For this reason storage of red cells in SAGM solution, supplemented with citrate, was tested and compared with storage in SAGM and CPD plasma. The results demonstrate a positive effect of citrate as an added ingredient in the SAGM solution. Significantly lower levels of primarily FPA but also decreased activities of plasma kallikrein and general proteolysis were noticed. The decreased citrate concentration in Red Blood Cells stored in SAGM protein poor medium must be considered to have a destabilizing influence on the coagulation and on other connected enzymatic systems.

The role of the fibrinolytic system in deep vein thrombosis.

Some aspects of the function of the fibrinolytic system have been investigated in 37 patients with a recent incident of symptomatic and confirmed deep vein thrombosis and compared with findings in 20 healthy persons. New specific methods to measure tissue plasminogen activator (t-PA) activity and antigen before and after venous occlusion and the recently discovered fast inhibitor to t-PA were employed. Thirteen of the patients with deep vein thrombosis (35%) had t-PA activity less than 0.5 IU/ml after venous occlusion, whereas the lowest activity found among the healthy individuals was 0.56 IU/ml. The t-PA inhibitor level in the total patient group was 3.8 +/- 3.7 U/ml (range 0 to 15.0 U/ml; median 2.9 U/ml) as compared with 0.7 +/- 0.7 U/ml in the healthy (median 0.5, range 0 to 2.4 U/ml). In the 13 patients with low t-PA activity in postocclusion plasma samples the inhibitor level was 6.0 +/- 4.4 U/ml. Furthermore, in this group of patients a significantly lesser release of t-PA antigen (3.7 +/- 2.8 micrograms/L) was found as compared with that in the healthy individuals (9.5 +/- 6.0 micrograms/L). Thus, two months after their first incident of symptomatic deep vein thrombosis many of the patients (35%) were found to have decreased fibrinolytic activity. This is the result of highly increased plasma levels of a novel fast inhibitor toward t-PA in combination with a poor ability to release t-PA. Possibly the decreased fibrinolytic activity did play a role in the pathogenesis of deep vein thrombosis in these patients.

A novel fast inhibitor to tissue plasminogen activator in plasma, which may be of great pathophysiological significance.

A novel, fast inhibitor to tissue plasminogen activator (t-PA) has been demonstrated in plasma samples. The inhibitor can be titrated in plasma. Upon addition of t-PA to plasma, an inactive complex is formed with an estimated rate constant of about 10(7)M-1s-1. The molecular weight of the complex between t-PA and the inhibitor has been determined as about 120,000. Thus, a molecular weight of about 55,000 would be expected for the inhibitor moiety in the complex. However, by gel filtration of plasma, rich in the inhibitor, an apparent molecular weight of about 200,000 was found. About 100,000 fold purification of the complex from plasma has been achieved by employing immuno adsorption chromatography (antibodies against porcine t-PA and monoclonal antibodies against human t-PA), ion-exchange chromatography and gel-filtration. Low concentrations of the new t-PA inhibitor were normally found in healthy individuals (0.7 +/- 0.7 U/ml). However, many patients with thrombosis or coronary heart disease had increased levels (3.8 +/- 3.7 U/ml and 2.8 +/- 2.2 U/ml, respectively). In normal pregnancy the concentration of the fast t-PA inhibitor increases linearly from week 10 to the time about term (5.1 +/- 0.9 U/ml). At present the origin as well as the physiological role of this novel t-PA inhibitor remains unclear. The increased levels observed in many patients with thrombotic diseases or in conditions frequently associated with thrombotic complications anticipates a role in the development of thrombosis. However, more work is needed to prove this hypothesis.

Inactivation of tissue plasminogen activator in plasma. Demonstration of a complex with a new rapid inhibitor.

A new specific and sensitive method for determination of tissue plasminogen activator (t-PA) in plasma samples has been used to demonstrate the presence of a fast inhibitor to t-PA in plasma. By adding [35S]Met internally labeled t-PA (Mr approximately 70,000) to plasma, we were able to demonstrate the rapid formation of a stable complex with an apparent molecular weight of about 115,000 as estimated by gel filtration. The complex was partially purified by immunoadsorbtion chromatography on insolubilized antibodies against porcine t-PA, and a molecular weight of about 120,000 was found by dodecyl sulfate-polyacrylamide gel electrophoresis. From the apparent molecular weight of the complex (120,000) and the molecular weight of t-PA (70,000), a molecular weight of about 50,000 would be expected for the inhibitor. However, gel filtration of inhibitor-rich plasma resulted in the appearance of a symmetrical peak of t-PA inhibitory activity with an apparent molecular weight of about 205,000. The reason for this discrepancy is not known, but several different models are possible.

Activation of blood coagulation, fibrinolytic and kallikrein systems during storage of plasma.

This study deals with the question of how blood coagulation, kallikrein and fibrinolytic systems are affected by storage of plasma at +6 degrees C. Blood was collected into citrate phosphate dextrose adenine (CPD) or acid citrate dextrose (ACD) and the plasma samples were stored at +6 degrees C for 35 days. Samples were taken at weekly intervals for assays of various parameters of the different systems. No significant changes were observed in the levels of the main thrombin inhibitor, antithrombin III. At the end of the storage period, however, fibrinopeptide A levels increased markedly, particularly in the ACD plasma, indicating thrombin activation. There was no change in the plasminogen level, but a decrease in the levels of antiplasmin and urokinase inhibitors and an increase in the level of the fibrinogen degradation fragment B beta 15-42 were observed, indicating activation of the fibrinolytic system. The level of antikallikrein activity decreased sharply in ACD plasma; CPD plasma was less affected. This decrease was parallel to the increase in spontaneous proteolytic activity and correlated with the increase in fibrinopeptide A. Prolonged storage of plasma of +6 degrees C thus resulted in the activation of coagulation, fibrinolytic and kallikrein systems and decrease in inhibitors. The activation was much more pronounced in ACD than in CPD plasma.

Fibrinolytic and coagulation mechanisms in stages of inflammation: a study of BCG-induced pleural exudate in guinea pig.

Pleural fluid from an early, active phase of BCG-induced pleurisy was compared with fluid from late, healing phase, characterized by fibrinous adhesions. Exudates were tested for proteolytic activity on chromogenic peptide substrates designed for plasmin, tissue plasminogen activator, factor Xa, thrombin and plasma kallikrein. Considerable activity of active-phase pleural fluid was found on all of these substrates, and significantly lower values in the healing phase. Most exudates from both stages had very low fibrinogen concentration. Fibrinopeptide A, fibrinolytic products and antiplasmin were found in all exudates. Little or no fibrinolytic effect of pleural fluid was demonstrable on plasminogen free fibrin plates, despite the high activities on the low molecular weight substrates. Occurrence of alpha 2-macroglobulin-enzyme complexes is suggested as an explanation. The experimental results indicate that protease of the fibrinolytic and coagulation systems are active in the chronic inflammation of pleurisy, with higher levels of activity in active pleurisy phase.