In vitro evaluation of aspiration of hyaluronic acid filler with a new saline flashing method.

AIMS: In vitro evaluation of aspiration of viscous hyaluronic acid (HA) fillers through dermatologic needles, with and without a new solution for safe aspiration with a saline flashing procedure. The final objective is to be able to offer an easy-to-use device for a practitioner who injects fillers under and into the skin blindly. This device aims to protect him from an immediate and reproducible intravascular venous or arterial injection whatever the type of product used and independently of its rheological properties or the diameter of the needle used. MATERIALS AND METHODS: We performed in vitro aspiration experiment using syringes and commonly used needle sizes (27G, 29G). We measured the time to observe a blood being aspirated through the needle with HA filler. Intervals longer than 10 seconds were considered as false results. We added a new approach of flashing a saline solution through the needle with a filler prior to aspiration. Saline solution pressurized by a syringe plunger opens a fluid path in the needle and enables the successful and efficient aspiration in 1-2 seconds through smallest needle size for 6 commercial HA fillers. CONCLUSION: The saline flashing of the dermatological needles with HA fillers appeared to be a feasible and efficient solution for the 100% safe aspiration safety test. RESULTS: Aspiration was possible only in few trials with HA fillers for the 27G and all false for 29G needle, respectively. The aspiration with the novel saline flashing procedure is successful for 100% of cases with the two commonly used HA fillers and all needle sizes. The time to aspiration is also significantly shorter with the saline flashing.

Heparan sulfate chains contribute to the anticoagulant milieu in malignant pleural effusion.

BACKGROUND: While malignant pleural effusion (MPE) is a common and significant cause of morbidity in patients with cancer, current treatment options are limited. Human heparanase, involved in angiogenesis and metastasis, cleaves heparan sulfate (HS) side chains on the cell surface. AIMS: To explore the coagulation milieu in MPE and infectious pleural effusion (IPE) focusing on the involvement of heparanase. METHODS: Samples of 30 patients with MPE and 44 patients with IPE were evaluated in comparison to those of 33 patients with transudate pleural effusions, using heparanase ELISA, heparanase procoagulant activity assay, thrombin and factor Xa chromogenic assays and thromboelastography. A cell proliferation assay was performed. EMT-6 breast cancer cells were injected to the pleural cavity of mice. A peptide inhibiting heparanase activity was administered subcutaneously. RESULTS: Levels of heparanase, factor Xa and thrombin were significantly higher in exudate than transudate. Thromboelastography detected almost no thrombus formation in the whole blood, mainly on MPE addition. This effect was completely reversed by bacterial heparinase. Direct measurement revealed high levels of HS chains in pleural effusions. Higher proliferation was observed in tumour cell lines incubated with exudate than with transudate and it was reduced when bacterial heparinase was added. The tumour size in the pleural cavity of mice treated with the heparanase inhibitor were significantly smaller compared with control (p=0.005). CONCLUSIONS: HS chains released by heparanase form an anticoagulant milieu in MPE, preventing local thrombosis and enabling tumour cell proliferation. Inhibition of heparanase might provide a therapeutic option for patients with recurrent MPE.

Heparanase Level in the Microcirculation as a Possible Modulator of the Metastatic Process.

Metastasis most commonly occurs in the liver, lung, bone, and brain, implying its preference for specific organs. We hypothesized that organ microcirculation coagulation environment predisposes to tumor cell retention. Coagulation factors were analyzed using immunostaining, enzyme-linked immunosorbent assay, and heparanase procoagulant activity assay. In normal mice, expression levels of heparanase, tissue factor (TF), TF pathway inhibitor (TFPI), and TFPI-2 were low in the microcirculation of the liver, lung, brain cortex, and bone, and high in the microcirculation of the subcutis, skeletal muscle, brain subcortex, and bone marrow. C57BL/6 mice injected s.c. with B16 (F10) melanoma cells demonstrated lower levels of heparanase, TF, TFPI, and TFPI-2 in metastasis blood vessels compared to those in the primary tumor. In these mice with metastasis, liver and lung microcirculation turned to express high levels of coagulation proteins. Additionally, although mice with heparanase overexpression developed a larger primary tumor, they did not demonstrate a tendency for metastasis, as opposed to controls (P < 0.0001). Human umbilical vein endothelial cells, incubated with the B16 melanoma cell medium for 2 hours, expressed decreased levels of heparanase, TF, TFPI, and TFPI-2, and the effect was reversed by a peptide-inhibiting heparanase/TF complex interaction (P < 0.001). In summary, metastasis has a predilection to organs with low levels of heparanase, TF, TFPI, and TFPI-2 in the microcirculation, which enables tumor cell retention. Heparanase has a role in regulating the microcirculation milieu.

Peptides inhibiting heparanase procoagulant activity significantly reduce tumour growth and vascularisation in a mouse model.

Heparanase is implicated in angiogenesis and tumour progression. We previously demonstrated that heparanase might also affect the haemostatic system in a non-enzymatic manner. It forms a complex and enhances the activity of the blood coagulation initiator tissue factor (TF). Peptides that we generated from TF pathway inhibitor (TFPI)-2, which inhibit heparanase procoagulant activity, were recently demonstrated to attenuate inflammation in a sepsis mouse model. The present study was designated to explore peptides effects on tumour growth and vascularisation. Cell lines of mouse melanoma (B16), mouse breast cancer (EMT-6), and human breast cancer (MDA-231) were injected subcutaneously to mice. Inhibitory peptides 5, 6 and 7 were injected subcutaneously in the area opposite to the tumour side. In the three tumour cell lines, peptides 5, 6 and 7 inhibited tumour growth and vascularisation in a dose-dependent manner, reaching a 2/3 reduction compared to control tumours (p<0.001). Additionally, a survival advantage (p<0.05) and reduced plasma thrombin-antithrombin complex (p<0.05) were observed in the treatment groups. Peptides delayed tumour relapse by six days and inhibited relapsed tumour size (p<0.001). In vitro, peptides did not inhibit tumour cell proliferation, migration or heparanase degradation of heparan sulfate chains, but significantly decreased tube formation. In conclusion, peptides inhibiting heparanase procoagulant activity significantly reduced tumour growth, vascularisation, and relapse. The procoagulant domain in heparanase protein may play a role in tumour growth, suggesting a new mechanism of coagulation system involvement in cancer.

JAK-2 V617F mutation increases heparanase procoagulant activity.

Patients with polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are at increased risk of arterial and venous thrombosis. In patients with ET a positive correlation was observed between JAK-2 V617F mutation, that facilitates erythropoietin receptor signalling, and thrombotic events, although the mechanism involved is not clear. We previously demonstrated that heparanase protein forms a complex and enhances the activity of the blood coagulation initiator tissue factor (TF) which leads to increased factor Xa production and subsequent activation of the coagulation system. The present study was aimed to evaluate heparanase procoagulant activity in myeloproliferative neoplasms. Forty bone marrow biopsies of patients with ET, PV, PMF and chronic myelogenous leukaemia (CML) were immunostained to heparanase, TF and TF pathway inhibitor (TFPI). Erythropoietin receptor positive cell lines U87 human glioma and MCF-7 human breast carcinoma were studied. Heparanase and TFPI staining were more prominent in ET, PV and PMF compared to CML. The strongest staining was in JAK-2 positive ET biopsies. Heparanase level and procoagulant activity were higher in U87 cells transfected to over express JAK-2 V617F mutation compared to control and the effect was reversed using JAK-2 inhibitors (Ruxolitinib, VZ3) and hydroxyurea, although the latter drug did not inhibit JAK-2 phosphorylation. Erythropoietin increased while JAK-2 inhibitors decreased the heparanase level and procoagulant activity in U87 and MCF-7 parental cells. In conclusion, JAK-2 is involved in heparanase up-regulation via the erythropoietin receptor. The present findings may potentially point to a new mechanism of thrombosis in JAK-2 positive ET patients.

Emotional memory formation under lower versus higher stress conditions.

An exposure to stress can enhance memory for emotionally arousing experiences. The phenomenon is suggested to be amygdala-dependent and in accordance with that view the amygdala was found to modulate mnemonic processes in other brain regions. Previously, we illustrated increased amygdala activation and reduced activation of CA1 following spatial learning under higher versus lower stress conditions. When spatial learning was followed by reversal training interference, impaired retention was detected only under higher stress condition. Here we further evaluate the potential implications of the difference in the level of amygdala activation on the quality of the memory formed under these stress conditions. We attempted to affect spatial memory consolidation under lower or higher stress conditions by either introducing a foot shock interference following massed training in the water maze; by manipulating the threshold for acquisition employing either brief (3 trials) or full (12 trials) training sessions; or by employing a spaced training (over 3 days) rather than massed training protocol. The current findings reveal that under heightened emotionality, the process of consolidation seems to become less effective and more vulnerable to interference; however, when memory consolidation is not interrupted, retention is improved. These differential effects might underlie the complex interactions of stress, and, particularly, of traumatic stress with memory formation processes.

Activation pattern of the limbic system following spatial learning under stress.

Anatomical evidence suggests an interplay between the dorsal CA1 of the hippocampus (CA1), the basolateral amygdala (BLA) and the entorhinal cortex (EC), but their specific interactions in the context of emotional memory remain obscure. Here, we sought to elucidate the activation pattern in these areas following spatial learning under different stress conditions in the Morris water maze, using cAMP response element-binding protein (CREB) activation as a marker. Stress levels were manipulated by maintaining the water maze at one of two different temperatures: lower stress (warm water) or higher stress (cold water). Three groups of animals were tested under each condition: a Learning group, trained in the water maze with a hidden escape platform; a No-Platform group, subjected to the maze without an escape platform; and a Naïve group. To evaluate the quality of the spatial memory formed, we also tested long-term memory retention of the initial location of the platform following an interference procedure (reversal training). In the CA1 and EC, we found different CREB activation patterns for the lower- and higher-stress groups. By contrast, in the BLA a similar pattern of activation was detected under both stress levels. The data reveal a difference in the sensitivity of the memory to interference, with reversal training interference affecting the memory of the initial platform location only under the higher-stress condition. The results suggest that stress-dependent alterations in limbic system activation patterns underlie differences in the quality of the memory formed.

Effect of fetal number on maternal serum uric acid concentration.

The aim of this study was to examine whether maternal serum uric acid (UA) concentrations are influenced by the number of fetuses and whether this effect is confounded by maternal body mass index (BMI). Medical records of 207 consecutive twin and 69 triplet pregnancies admitted to our high-risk pregnancy unit between 1994 and 1998 were reviewed. Pregnancies complicated by acute or chronic renal diseases, vascular diseases, hypertension, hemolysis, diabetes mellitus, or proteinuria were excluded. The remaining 137 twin and 42 triplet pregnancies were matched with 118 consecutive singleton pregnancies who met the same exclusion criteria and were admitted in the first half of 1998. Each birth order study group was further stratified and compared within three maternal BMI subgroups. Serum UA concentrations were higher in twin and triplet pregnancies compared to singletons (4.6 +/- 1.3, 5.2 +/- 1.2, and 3.8 +/- 0.7 mg%, respectively; p <0.01). These differences in UA concentration persisted after grouping by BMI classes. Serum UA concentration in pregnancy are positively correlated with the number of fetuses. For clinical purpose, the UA cutoff concentration should be adjusted using mean + 2 SD as follows: 5.2, 7.2, and 7.6 for singleton, twin, and triplets, respectively.