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The urokinase-type plasminogen activator (uPA) is a serine protease involved in fibrinolysis, tissue remodeling, angiogenesis, and cancer metastasis. This dissertation investigated the factors that affect the activity of this enzyme beyond the three amino acids that constitute the catalytic triad. In Chapter 2, a new method to purify single-chain uPA from inclusion bodies was developed. This method increased the time of storage of single-chain uPA to 2 months. In Chapter 3, the effect of uPA receptor (uPAR) binding on the protease activity of uPA was studied. I showed that monomeric uPAR does not affect the catalytic activity of uPA in solution. In Chapter 4, the ability of single-chain uPA to autoactivate was demonstrated. Two-chain uPA can cleave single-chain uPA after K135 in the disordered part of the light chain and after K158. Only this last cleavage causes activation of uPA. In addition, hydrogen deuterium exchange mass spectrometry (HDX-MS) was used to characterize the dynamic differences between the uPA zymogen and its active form. A mechanism was proposed in which the dimerization of the uPAR could promote autoactivation of single-chain uPA on cell surfaces. In Chapter 5, the effect of the amino terminal fragment (ATF) on the protease domain of uPA was evaluated. Full-length uPA was faster at catalyzing the hydrolysis of plasminogen than the form of uPA that was missing the ATF. Surprisingly, the behavior was opposite for a small chromogenic substrate. In addition, full-length uPA also had less affinity for a small substrate inhibitor than the protease domain alone form. HDX-MS revealed that when ATF is present, the deuterium uptake of the 8-strand that is located below the S1 specificity pocket is reduced. In Chapter 6, the effect of the disordered part of the light chain on the protease domain of murine uPA was assessed. The light chain increases the activity of the protease domain 5-fold and key interactions between the light-chain and the protease dom

Objective To identify serum proteins associated with MS and affected by interferon beta treatment. Methods Plasma samples from 29 untreated relapsing-remitting MS patients and 15 healthy controls were investigated with a multiplexed panel containing 92 proteins related to inflammation. Follow-up samples were available from 13 patients at 1 and 3 months after initiation of treatment with interferon beta-1a. Results Ten proteins were differentially expressed in MS patients. Five of these were altered by treatment with IFN-β 1a: uPA, CX3CL1, CCL2, TRAIL and IL18. Conclusion CCL2 and TRAIL were confirmed to be modulated with interferon beta treatment in MS. As novel findings, we now report that uPA and CX3CL1 were differentially expressed in MS and increased after IFN-beta-1a treatment. Conflicting results have been reported on how interferon beta affects IL-18., Title in Web of Science: Urokinase, CX3CL1, CCL2, TRAIL and IL-18 induced by interferon-beta treatment

Objective To identify serum proteins associated with MS and affected by interferon beta treatment. Methods Plasma samples from 29 untreated relapsing-remitting MS patients and 15 healthy controls were investigated with a multiplexed panel containing 92 proteins related to inflammation. Follow-up samples were available from 13 patients at 1 and 3 months after initiation of treatment with interferon beta-1a. Results Ten proteins were differentially expressed in MS patients. Five of these were altered by treatment with IFN-β 1a: uPA, CX3CL1, CCL2, TRAIL and IL18. Conclusion CCL2 and TRAIL were confirmed to be modulated with interferon beta treatment in MS. As novel findings, we now report that uPA and CX3CL1 were differentially expressed in MS and increased after IFN-beta-1a treatment. Conflicting results have been reported on how interferon beta affects IL-18., Title in Web of Science: Urokinase, CX3CL1, CCL2, TRAIL and IL-18 induced by interferon-beta treatment

Objective To identify serum proteins associated with MS and affected by interferon beta treatment. Methods Plasma samples from 29 untreated relapsing-remitting MS patients and 15 healthy controls were investigated with a multiplexed panel containing 92 proteins related to inflammation. Follow-up samples were available from 13 patients at 1 and 3 months after initiation of treatment with interferon beta-1a. Results Ten proteins were differentially expressed in MS patients. Five of these were altered by treatment with IFN-β 1a: uPA, CX3CL1, CCL2, TRAIL and IL18. Conclusion CCL2 and TRAIL were confirmed to be modulated with interferon beta treatment in MS. As novel findings, we now report that uPA and CX3CL1 were differentially expressed in MS and increased after IFN-beta-1a treatment. Conflicting results have been reported on how interferon beta affects IL-18., Title in Web of Science: Urokinase, CX3CL1, CCL2, TRAIL and IL-18 induced by interferon-beta treatment

La présence d’un biofilm dans le cathéter de dialyse péritonéale où les bactéries sont encapsulées, inaccessibles au traitement antibiotique, et relarguées régulièrement, à bas bruit, dans le dialysat, explique le mieux le caractère récidivant d’une péritonite infectieuse à l’arrêt de l’antibiothérapie, chez des patients en dialyse péritonéale. Nous rapportons ici quatre cas cliniques où l’injection d’urokinase dans le cathéter de dialyse péritonéale, en complément du traitement antibiotique, a permis d’éradiquer une péritonite récidivante à Staphylocoque epidermidis dans deux cas, à Acinetobacter johnsonii dans un cas et à Staphylocoque haemolyticus dans un cas. Ce traitement adjuvant, dénué d’effets secondaires, a permis la guérison de l’épisode infectieux, d’éviter l’ablation du cathéter de dialyse péritonéale et le transfert en hémodialyse, chez ces patients en dialyse péritonéale présentant une péritonite récidivante., [Peritoneal dialysis catheter sterilization by urokinase administration in case of relapsing peritonitis: About four observations] The presence of a biofilm within the peritoneal dialysis catheter where bacteria are encapsulated, protected from the action of antibiotics and insidiously liberated within the dialysate, best explains the relapse of the infectious peritonitis, when antibiotics are withdrawn. We here report a serie of four clinical cases in whom the administration of urokinase within the peritoneal catheter in addition to the current antibiotherapy, has cured relapsing peritonitis due to Staphylococcus epidermidis in two cases, Acinetobacterjohnsonii in one case and Staphylococcus haemolyticus in one case, respectively. This approach, safe and easy, allowed the infection eradication and did prevent a catheter removal and a potential transfer of the patients to hemodialysis.

Background: Hereditary palmoplantar keratodermas (PPK) represent a heterogeneous group of rare skin disorders with epidermal hyperkeratosis of the palms and soles, with occasional additional manifestations in other tissues. Mutations in at least 69 genes have been implicated in PPK, but further novel candidate genes and mutations are still to be found. Objectives: To identify mutations underlying PPK in a cohort of 64 patients. Methods: DNA of 48 patients was analysed on a custom-designed in-house panel for 35 PPK genes, and 16 patients were investigated by a diagnostic genetic laboratory either by whole-exome sequencing, gene panels or targeted single-gene sequencing. Results: Of the 64 PPK patients, 32 had diffuse (50%), 19 focal (30%) and 13 punctate (20%) PPK. None had striate PPK. Pathogenic mutations in altogether five genes were identified in 31 of 64 (48%) patients, the majority (22/31) with diffuse PPK. Of them, 11 had a mutation in AQP5, five in SERPINB7, four in KRT9 and two in SLURP1. AAGAB mutations were found in nine punctate PPK patients. New mutations were identified in KRT9 and AAGAB. No pathogenic mutations were detected in focal PPK. Variants of uncertain significance (VUS) in PPK-associated and other genes were observed in 21 patients that might explain their PPK. No suggestive pathogenic variants were found for 12 patients. Conclusions: Diffuse PPK was the most common (50%) and striate PPK was not observed. We identified pathogenic mutations in 48% of our PPK patients, mainly in five genes: AQP5, AAGAB, KRT9, SERPINB7 and SLURP1., QC 20220314

Background: There is evidence of increased low grade inflammation (LGI) in schizophrenia patients. However, the inter-individual variation is large and the association with demographic, somatic and psychiatric factors remains unclear. Our aim was to explore whether levels of the novel LGI marker soluble urokinase plasminogen activator receptor (suPAR) were associated with clinical factors in schizophrenia and if such associations were sex-dependent. Method: In this observational study a total of 187 participants with schizophrenia (108 males, 79 females) underwent physical examination and assessment with clinical interviews (Positive and Negative Syndrome Scale (PANSS), Calgary Depression Scale for Schizophrenia (CDSS), Alcohol Use Disorder Identification Test (AUDIT), and Drug Use Disorder Identification Test (DUDIT)). Blood levels of suPAR, glucose, lipids, and high sensitivity C-reactive protein (hsCRP) were determined and body mass index (BMI) calculated. Multivariable linear regression analyses were used adjusting for confounders, and sex interaction tested in significant variables. Results: Adjusting for sex, age, current tobacco smoking and BMI, we found that levels of hsCRP and depressive symptoms (CDSS) were positively associated with levels of suPAR (p < 0.001). The association between suPAR and CDSS score was significant in females (p < 0.001) but not in males. Immune activation measured by hsCRP was not associated with depressive symptoms after adjusting for BMI. Conclusion: Our findings indicate that increased suPAR levels are associated with depressive symptoms in females with schizophrenia, suggesting aberrant immune activation in this subgroup. Our results warrant further studies, including longitudinal follow-up of suPAR levels in schizophrenia and experimental studies of mechanisms.

Background: There is evidence of increased low grade inflammation (LGI) in schizophrenia patients. However, the inter-individual variation is large and the association with demographic, somatic and psychiatric factors remains unclear. Our aim was to explore whether levels of the novel LGI marker soluble urokinase plasminogen activator receptor (suPAR) were associated with clinical factors in schizophrenia and if such associations were sex-dependent. Method: In this observational study a total of 187 participants with schizophrenia (108 males, 79 females) underwent physical examination and assessment with clinical interviews (Positive and Negative Syndrome Scale (PANSS), Calgary Depression Scale for Schizophrenia (CDSS), Alcohol Use Disorder Identification Test (AUDIT), and Drug Use Disorder Identification Test (DUDIT)). Blood levels of suPAR, glucose, lipids, and high sensitivity C-reactive protein (hsCRP) were determined and body mass index (BMI) calculated. Multivariable linear regression analyses were used adjusting for confounders, and sex interaction tested in significant variables. Results: Adjusting for sex, age, current tobacco smoking and BMI, we found that levels of hsCRP and depressive symptoms (CDSS) were positively associated with levels of suPAR (p < 0.001). The association between suPAR and CDSS score was significant in females (p < 0.001) but not in males. Immune activation measured by hsCRP was not associated with depressive symptoms after adjusting for BMI. Conclusion: Our findings indicate that increased suPAR levels are associated with depressive symptoms in females with schizophrenia, suggesting aberrant immune activation in this subgroup. Our results warrant further studies, including longitudinal follow-up of suPAR levels in schizophrenia and experimental studies of mechanisms.

Rheumatoid arthritis (RA) is a chronic inflammatory disease of synovial joints. Accumulating evidence implicates the pathogenic role of the urokinase plasminogen activation system (uPAS) in RA and experimental arthritis models. Collagen-induced arthritis (CIA) is one of the most widely used and clinically relevant models of RA for the evaluation of novel anti-arthritic therapeutic candidates. Urokinase plasminogen activator-directed monoclonal antibodies have shown ameliorating effects in a mouse model of CIA. Previous efforts in our laboratory produced novel amiloride analogues as inhibitors of human uPA with improved selectivity over other trypsin-like serine proteases (TLSPs). The lead compound, BB2-30F, completely eliminated metastasis in uPA-driven mouse xenograft cancer models, suggesting the potential for its use in inflammatory disease models such as RA where uPAS is upregulated. As evident from the literature, the CIA model in rats more closely recapitulates human RA than equivalent models in mice. This motivated our selection of rat CIA for testing the effects of BB2-30F in this disease model. However, the uPAS has not been characterised in rat CIA. Thus, the overall objectives of the study were to characterise uPAS in female Lewis rat CIA and then to investigate the in vivo efficacy of BB2-30F compared to the standard of care treatment methotrexate (MTx).

BackgroundDiabetic nephropathy is a common diabetes mellitus complication associated with hypertension, proteinuria, and excretion of urinary plasmin that activates the epithelial sodium channel, ENaC, in vitro. Here we hypothesized that the deletion of plasminogen and amiloride treatment protect against hypertension in diabetes mellitus.Methods and ResultsMale plasminogen knockout (plasminogen-deficient [Plg(-/-)]) and wild-type mice were rendered diabetic with streptozotocin. Arterial blood pressure was recorded continuously by indwelling catheters before and during 10 days of angiotensin II infusion (ANGII; 30-60 ng/kg per minute). The effect of amiloride infusion (2 mg/kg per day, 4 days) was tested in wild-type, diabetic ANGII-treated mice. Streptozotocin increased plasma and urine glucose concentrations and 24-hour urine albumin and plasminogen excretion. Diabetic Plg(-/-) mice displayed larger baseline albuminuria and absence of urine plasminogen. Baseline mean arterial blood pressure did not differ between groups. Although ANGII elevated blood pressure in wild-type, diabetic wild-type, and Plg(-/-) control mice, ANGII did not change blood pressure in diabetic Plg(-/-) mice. Compared with ANGII infusion alone, wild-type ANGII-infused diabetic mice showed blood pressure reduction upon amiloride treatment. There was no difference in plasma renin, ANGII, aldosterone, tissue prorenin receptor, renal inflammation, and fibrosis between groups. Urine from wild-type mice evoked larger amiloride-sensitive current than urine from Plg(-/-) mice with or without diabetes mellitus. Full-length gamma-ENaC and alpha-ENaC subunit abundances were not changed in kidney homogenates, but the 70 kDa gamma-ENaC cleavage product was increased in diabetic versus nondiabetic mice.ConclusionsPlasmin promotes hypertension in diabetes mellitus with albuminuria likely through the epithelial sodium channel.

BackgroundDiabetic nephropathy is a common diabetes mellitus complication associated with hypertension, proteinuria, and excretion of urinary plasmin that activates the epithelial sodium channel, ENaC, in vitro. Here we hypothesized that the deletion of plasminogen and amiloride treatment protect against hypertension in diabetes mellitus.Methods and ResultsMale plasminogen knockout (plasminogen-deficient [Plg(-/-)]) and wild-type mice were rendered diabetic with streptozotocin. Arterial blood pressure was recorded continuously by indwelling catheters before and during 10 days of angiotensin II infusion (ANGII; 30-60 ng/kg per minute). The effect of amiloride infusion (2 mg/kg per day, 4 days) was tested in wild-type, diabetic ANGII-treated mice. Streptozotocin increased plasma and urine glucose concentrations and 24-hour urine albumin and plasminogen excretion. Diabetic Plg(-/-) mice displayed larger baseline albuminuria and absence of urine plasminogen. Baseline mean arterial blood pressure did not differ between groups. Although ANGII elevated blood pressure in wild-type, diabetic wild-type, and Plg(-/-) control mice, ANGII did not change blood pressure in diabetic Plg(-/-) mice. Compared with ANGII infusion alone, wild-type ANGII-infused diabetic mice showed blood pressure reduction upon amiloride treatment. There was no difference in plasma renin, ANGII, aldosterone, tissue prorenin receptor, renal inflammation, and fibrosis between groups. Urine from wild-type mice evoked larger amiloride-sensitive current than urine from Plg(-/-) mice with or without diabetes mellitus. Full-length gamma-ENaC and alpha-ENaC subunit abundances were not changed in kidney homogenates, but the 70 kDa gamma-ENaC cleavage product was increased in diabetic versus nondiabetic mice.ConclusionsPlasmin promotes hypertension in diabetes mellitus with albuminuria likely through the epithelial sodium channel.

Mezenhimske matične ćelije (MMĆ) deo su populacije multipotentnih matičnih ćelija prisutnih u mnogim adultnim tkivima gde učestvuju u održavanju homeostaze. Zahvaljujući sposobnosti samoobnove, potencijalu diferenciranja u specijalizovane ćelije mezodermskog porekla i imunomodultatornim svojstvima, ove nediferencirane ćelije karakteriše širok opseg potencijalne terapijske primene. Regenerativnom kapacitetu MMĆ doprinosi i sposobnost migracije do oštećenih tkiva gde učestvuju u njihovom obnavlјanju što ove ćelije čini pogodnim za ćelijsku terapiju. Mezenhimske MĆ su do sada izolovane iz velikog broja adultnih tkiva i mnoge studije potvrdile su da njihov regenerativni potencijal u velikoj meri zavisi od izvora iz kojeg potiču. Prvobitno su izolovane iz kostne srži koja je do sada najčešće korišćen izvor MMĆ, međutim zbog invazivnih metoda izolacije i malog prinosa MMĆ, stvorena je potreba za alternativnim izvorima. Činjenica da su MMĆ izolovane i iz periferne krvi jedno je od značajnih otkrića iz ove oblasti i smatra se da ove ćelije najverovatnije vode poreklo iz kostne srži, međutim nije dovoljno poznato na koji način migriraju i bivaju privučene u oštećena tkiva. Poslednjih godina se kao povolјan izvor MMĆ sa potencijalnom primenom u regenerativnoj medicini sve češće pominju i perinatalna tkiva (pupčanik i placenta). Prednost pupčanika kao izvora MMĆ ogleda se u lakoj dostupnosti tkiva koje se smatra medicinskim otpadom, neinvazivnoj izolaciji ćelija i činjenici da su izolovane MMĆ po kapacitetu samoobnove i većoj ekspresiji markera pluripotentnost slične embrionalnim MĆ. Iako se već nekoliko decenija MMĆ intenzivno istražuju, uprkos velikom doprinosu nauci i medicini, o njihovom upkupnom terapijskom potencijalu se još uvek nedovoljno zna. Trenutno nije poznat univerzalan mehanizam terapijskog delovanja MMĆ, a mnogobrojni nalazi sve više u poslednje vreme upućuju da ponašanje i uloge MMĆ zavise od konteksta mikrosredine u kojoj obitavaju i/ili deluju, kao mestu int, present in many adult tissues where they participate in maintaining of homeostasis. Due to their self-renewal capacity, differentiation potential into specialized cells of mesodermal origin and immunomodulatory features, these undifferentiated cells are characterized with wide range of potential therapeutic applications. Their ability to migrate towards damaged tissues where they participate in tissue repair, also contributes to their regenerative potential which makes them suitable for cell therapy. MSCs have so far been isolated from various adult tissues and many studies confirmed that MSCs’ regenerative potential depends largely on the suorce which they originate from. They were originaly isolated from bone marrow which has been the most commonly used source of MSCs so far, however due to invasive methods of isolation and low MSCs yield, there is a need for alternative sources of MSCs. The fact that these cells were isolated from peripheral blood is one of the significant discoveries in this area and it is believed that these MSCs are most likely to originate from bone marrow, but how they migrate and home to damaged tissues has not yet been elucidated completely. Perinatal tissues (umbilical cord and placenta) have been recently increasingly suggested as favourable sources of MSCs with potential applications in regenerative medicine. The advantage of umbilical cord as a source of MSCs is reflected in easy availability of tissue considered as medical waste, non-invasive cell isolation methods, and the fact that MSCs isolated from umbilical cord are similar to embryonic stem cells in the terms of self-renewal capacity and pluripotency markers’ expression. Eventhough MSCs have been extensively investigated for several decades, and despite their great contribution to science and medicine, their overall therapeutic potential is still poorly understood. Currently, there is no universal mechanism of MSCs’ therapeutic action known, and many findings have recently sugge

Background: Urokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion. Methods: Mouse uPAR was stably overexpressed in the mouse OSCC cell line AT84. The ratio of full-length versus cleaved uPAR as analysed by Western blotting and its regulation was assessed by addition of different protease inhibitors and transforming growth factor – β1 (TGF-β1). The role of uPAR cleavage in cell proliferation and migration was analysed using real-time cell analysis and invasion was assessed using the myoma invasion model. Results: We found that when uPAR was overexpressed a proportion of the receptor was cleaved, thus the cells presented both full-length uPAR and uPAR (II-III). Cleavage was mainly performed by serine proteases and urokinase plasminogen activator (uPA) in particular. When the OSCC cells were stimulated with TGF-β1, the production of the uPA inhibitor PAI-1 was increased, resulting in a reduction of uPAR cleavage. By inhibiting cleavage of uPAR, cell migration was reduced, and by inhibiting uPA activity, invasion was reduced. We could also show that medium containing soluble uPAR (suPAR), and cleaved soluble uPAR (suPAR (II-III)), induced migration in OSCC cells with low endogenous levels of uPAR. Conclus

Introduction: The prophylactic use of recombinant tissue plasminogen activator once weekly reduces the incidence rate of tunneled cuffed catheter (TCC) malfunction and bacteremia as compared to the exclusive use of heparin as locking solution. Restricting the use of prophylactic thrombolytic agents to patients with a history of thrombotic TCC malfunction could be more cost effective. We conduct a multicenter, double-blind, randomized controlled trial and test the hypothesis that weekly use of urokinase lock will reduce the incidence of thrombotic malfunction by 50% in prevalent hemodialysis patients with a history of thrombotic malfunction. Methods: Patients with a history of at least two separate TCC thrombotic dysfunctions treated with urokinase lock during the 6 months preceding inclusion are recruited in eight Belgian dialysis units. Patients are randomized in two groups: the control group receiving Taurolock™-HEP500 (heparin 500 IU/mL, taurolidine, citrate 4%) after each hemodialysis session and the treatment group receiving Taurolock-U 25,000 (urokinase 25,000, taurolidine, citrate 4%) once a week and the standard Taurolock-HEP500 at the end of the two others sessions. The primary outcome is the incidence rate of TCC thrombotic dysfunction defined by the use of urokinase. The secondary outcomes are the incidence rate of TCC removal and systemic thrombolysis. For the study, both patients and healthcare staff are blinded to treatment allocation. Conclusions: The present trial is the first to investigate the effect of Taurolock-U 25,000 catheter lock once a week as secondary prevention in hemodialysis patients with the highest risk of TCC-related thrombotic dysfunction., SCOPUS: ar.j, info:eu-repo/semantics/published

Abstract Two isomeric piperidine derivatives (meta and para isomers) were used as arginine mimics in the P1 position of a cyclic peptidic inhibitor (CPAYSRYLDC) of urokinase-type plasminogen activator. The two resulting cyclic peptides showed vastly different affinities (∼70 fold) to the target enzyme. X-ray crystal structure analysis showed that the two P1 residues were inserted into the S1 specificity pocket in indistinguishable manners. However, the rest of the peptides bound in entirely different ways on the surface of the enzyme, and the two peptides have different conformations, despite the highly similar sequence. These results demonstrate how the subtle difference in P1 residue can dictate the exosite interactions and the potencies of peptidic inhibitors, and highlight the importance of the P1 residue for protease inhibition. This study provides important information for the development of peptidic agents for pharmacological intervention.

Purpose – to compare the efficacy of urokinase and urokinase combined with low molecular weight heparin (Cibor 2500 mg) for the treatment of critical limb ischemia in diabetic foot.Material and methods. The first group of studies (IG 1) – 39 patients, received 1,0–0,05 million I U/day f or 20–26 days urokinase along with treatment protocol. The second group (IG 2) – 19 patients received treatment similar to one IG but while taking Cibor. Control Group (CG, 11 patients) recruited retrospectively. It was compared the dynamics of level of fibrinogen, TcPO2 and dynamics of ulcer healing (assessed by the beginning of treatment, at 10 and 20 days). Results. In both experimental groups dynamic of fibrinogen was statistically better than in the CG (p < 0,01) – its level reduced in an IG 1 and 2 f rom (4.55 ± 1.64) g/l and (4.82 ± 1.10) g/l to (2.43 ± 1.03) g/l and (2.57 ± 0.82) g/l after 20 days respectively. In CG level of fibrinogen decreased from (4.70 ± 1.18) g/l to (3.93 ± 1.01) g/l. There was no significant difference in the dynamic of fibrinogen between 1 and 2 IG. Just significantly (p <0.01) improved level of TcPO2 1 and 2 IG (f rom (10.36 ± 3.02) and (9.21 ± 3.05) mm Hg on the beginning to (19.26 ± 6.54) and (23.65 ± 3.12) mm Hg . at the end of treatment ) compared with the dynamics in the CG ((13. 50 ± 2.66) mm Hg at beginning and (14.80 ± 2.71) mm Hg at the end of treatment) . But with the additional use Cibor TcPO2 was statistically better on 10th ((15.32 ± 3.41) mm Hg vs (13.38 ± 3.36) mm Hg . ). and on the twentieth day. In the hospital after stabilizing among patients of IG 1 13 (33.33%) amputations were performed, including 3 – at the level of the famur, 3 – at the level of the shin, 7 – at the level of the foot. In IG 2 6 (31.58%) patients were perf ormed amputations , including 1- at lev el of the lower third of the femur, 1 – at the level of the shin, 4 – foot amputation level. In CG conducted 7 (64%), including 2 – at hip level, 2 – at the level o, Цель – сравнить эффективность применения урокиназы и комбинации урокиназы снизкомолекулярным гепарином (Цибор 2500 мг) для лечения критической ишемии при синдроме диабетической стопы.Материал и методы. В первую группу исследования (ОГ 1) вошли 39 больных, которые, кроме стандартного лечения, получали 1,00–0,05 млн МЕ/сутки урокиназы в течение 20–26 дней, во вторую группу (ОГ 2) – 19 больных, которые получали лечение, аналогичное таковому в ОГ 1, но на фоне приема Цибора. Группа контроля (КГ, 11 больных) набрана ретроспективно. Оценивали динамику содержания фибриногена, парциального давления кислорода в тканях нижних конечностей (TcPO2) и заживления язв (оценку проводили в начале лечения, через 10 и 20 дней). Результаты. Динамика уровня фибриногена в опытных группах была статистически лучше, чем в КГ (р<0,01) : он уменьшился через 20 дней в ОГ 1 и ОГ 2 с (4,55±1,64) и (4,82±1,10) г/л в начале лечения до (2,43±1,03) и (2,57±0,82) г/л соответственно, а в КГ – с (4,70±1,18) до (3,93±1,01) г/л. Не выявлено существенной разницы в динамике уровня фибриногена между ОГ 1 и ОГ 2 через 10 и 20 дней – (2,96±1,31) и (1,94±0,31) г/л соответственно. Также достоверно (р<0,01) улучшился уровень TcPO2 в ОГ 1 и ОГ 2 (с (10,36±3,02) и (9,21±3,05) мм рт. ст. в начале лечения до (19,26±6,54) и (23,65±3,12) мм рт. ст. в конце лечения) по сравнению с КГ ((13,50±2,66) и (14,80±2,71) мм рт. ст.). Однако на фоне применения Цибора уровень TcPO2 был статистически лучше через 10 и 20 дней. В стационарных условиях, после стабилизации состояния пациентов в ОГ 1 выполнено 13 (33,33%) ампутаций, из них 3 – на уровне бедра, 3 – на уровне голени, 7 – на уровне стопы. В ОГ 2 выполнены ампутации 6 (31,58%) больным, из них 1 – на уровне нижней трети бедра, 1 – на уровне голени, 4 – на уровне стопы. В КГ 7 (64%) больным проведены ампутации, из них 2 – на уровне бедра, 2 – на уровне голени, 3 – на уровне стопы.Выводы. Доказана эффективность сочетания урокиназы с низкомолекулярными гепаринамиотносител, Мета – порiвняти ефективнiсть застосування урокiнази та комбiнацi ї урокiнази з низькомолекулярним гепарином (Цибор 2500 мг) для лiкування критичної iшемiї за синдрому дiабетичної стопи. Матерiал та методи. До першої групи дослiдження (ДГ 1) залучено 39 хворих, якi, крiм стандартного лiкування, отримували 1,00–0,05 млн МО/добу урокiнази протягом 20–26 днiв, до другої групи (ДГ 2) – 19 хворих, якi отримували лiкування, аналогiчне такому у ДГ 1, але на тлi прийому Цибору. Групу контролю (КГ, 11 хворих) набрано ретроспективно. Оцiнювали динамiку вмiсту фiбриногену, парцiального тиску кисню в тканинах нижнiх кiнцiвок (TcPO2) i загоювання виразок (оцiнку проводили на початку лiкування, через 10 та 20 днiв).Результати. Динамiка рiвня фiбриногену у дослiдних групах була статистично кращою, нiж у КГ (р<0,01): вiн зменшився через 20 днiв в ДГ 1 та ДГ 2 з (4,55±1,64) та (4,82±1,10) г/л на початку лiкування до (2,43±1,03) та (2,57±0,82) г/л вiдповiдно, а в КГ – з (4,70±1,18) до (3,93±1,01) г/л. Не виявлено суттєвої рiзницi в динамiцi рiвня фiбриногену мiж ДГ 1 та ДГ 2 через 10 та 20 днiв – (2,96±1,31) та (1,94±0,31) г/л вiдповiдно. Так само достовiрно (р<0,01) полiпшився TcPO2 у ДГ 1 та ДГ 2 (з (10,36±3,02) та (9,21±3,05) мм рт. ст. на початку лiкування до (19,26±6,54) i (23,65±3,12) мм рт. ст. у кiнцi лiкування) порiвняно з КГ ((13,50±2,66) та (14,80±2,71) мм рт. ст.). Однак на тлi застосування Цибору TcPO2 був статистично кращим через 10 та 20 днiв. У стацiонарних умовах, пiсля стабiлiзацiї стану пацiєнтiв в 1ДГ виконано 13 (33,33%) ампутацiй, з них 3 – на рiвнi стегна, 3 – на рiвнi гомiлки, 7 – на рiвнi стопи. У ДГ 2 виконано ампутацiї 6 (31,58%) хворим, з них 1 – на рiвнi нижньої третини стегна, 1 – на рiвнi гомiлцi, 4 – на рiвнi стопи. У КГ 7 (64%) хворим проведено ампутацiї, з них 2 – на рiвнi стегна, 2 – на рiвнi гомiлки, 3 – на рiвнi стопи. Висновки. Доведено ефективнiсть поєднання урокiнази з низькомолекулярними гепаринами щодо загоєння виразкових дефектiв у хво

Background: Endovascular or intra-arterial treatment (IAT) increases the likelihood of recanalization in patients with acute ischemic stroke caused by a proximal intracranial arterial occlusion. However, a beneficial effect of IAT on functional recovery in patients with acute ischemic stroke remains unproven. The aim of this study is to assess the effect of IAT on functional outcome in patients with acute ischemic stroke. Additionally, we aim to assess the safety of IAT, and the effect on recanalization of different mechanical treatment modalities. Methods/design: A multicenter randomized clinical trial with blinded outcome assessment. The active comparison is IAT versus no IAT. IAT may consist of intra-arterial thrombolysis with alteplase or urokinase, mechanical treatment or both. Mechanical treatment refers to retraction, aspiration, sonolysis, or use of a retrievable stent (stent-retriever). Patients with a relevant intracranial proximal arterial occlusion of the anterior circulation, who can be treated within 6 hours after stroke onset, are eligible. Treatment effect will be estimated w

Plasminogen (Pg), the precursor of the proteolytic and fibrinolytic enzyme of blood, is converted to the active enzyme plasmin (Pm) by different plasminogen activators (tissue plasminogen activators and urokinase), including the bacterial activators streptokinase and staphylokinase, which activate Pg to Pm and thus are used clinically for thrombolysis. The identification of Pg-activators is therefore an important step in understanding their functional mechanism and derives new therapies.

64Cu-DOTA-AE105 is a novel positron emission tomography (PET) tracer specific to the human urokinase-type plasminogen activator receptor (uPAR). In preparation of using this tracer in humans, as a new promising method to distinguish between indolent and aggressive cancers, we have performed PET studies in mice to evaluate the in vivo biodistribution and estimate human dosimetry of 64Cu-DOTA-AE105. MethodsFive mice received iv tail injection of 64Cu-DOTA-AE105 and were PET/CT scanned 1, 4.5 and 22h post injection. Volume-of-interest (VOI) were manually drawn on the following organs: heart, lung, liver, kidney, spleen, intestine, muscle, bone and bladder. The activity concentrations in the mentioned organs [%ID/g] were used for the dosimetry calculation. The %ID/g of each organ at 1, 4.5 and 22h was scaled to human value based on a difference between organ and body weights. The scaled values were then exported to OLINDA software for computation of the human absorbed doses. The residence times as well as effective dose equivalent for male and female could be obtained for each organ. To validate this approach, of human projection using mouse data, five mice received iv tail injection of another 64Cu-DOTA peptide-based tracer, 64Cu-DOTA-TATE, and underwent same procedure as just described. The human dosimetry estimates were then compared with observed human dosimetry estimate recently found in a first-in-man study using 64Cu-DOTA-TATE. ResultsHuman estimates of 64Cu-DOTA-AE105 revealed the heart wall to receive the highest dose (0.0918mSv/MBq) followed by the liver (0.0815mSv/MBq), All other organs/tissue were estimated to receive doses in the range of 0.02–0.04mSv/MBq. The mean effective whole-body dose of 64Cu-DOTA-AE105 was estimated to be 0.0317mSv/MBq. Relatively good correlation between human predicted and observed dosimetry estimates for 64Cu-DOTA-TATE was found. Importantly

Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus (GAS), a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator (uPA) contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase (Ska) was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA−/− mice. The observed decrease in virulence in uPA−/−mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.

Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus (GAS), a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator (uPA) contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase (Ska) was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA−/− mice. The observed decrease in virulence in uPA−/−mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.

The paper determines the kinetic parameters of plasmins from eight species of mammals and their N-terminals. They were purified by the same methods (affinity chromatography and ion exchange) and activated with urokinase, starting from a common concentration and its kinetics judged according to Lineweaver-Burk coordinates. For such purpose, MichaelisMenten enzyme kinetics, which is widely used in the study of enzymes, was implemented. When compared with the molecular weight standard used, all plasminogen showed a purity exceeding 95% and a 92 kDa band on electrophoresis. Equine and canine plasmins showed the same KM due to the chromogenic substrate (0.438 mM), this being the one with the highest affinity in this study, and the human being the one with the lower affinity (5.3 mM). The catalytic constant and the conversion rate of the chromogenic substrate to product were also determined. The N-terminals of the plasminogens of the eight species were determined, and differences were found between humans and animals, as well as between some animals. Only pigs and sheep showed no differences in their N-terminal (DPPDDY). The unification of the method for purification of plasminogens for any species was demonstrated, as well as the kinetic differences of the eight plasmins studied and the similarities and differences in the sequence the N-terminals of the eight species., El artigo determina os parâmetros cinéticos das plasminas de oito espécies de mamíferos e seus terminais-N. Estes foram purificados pelos mesmos métodos (cromatografias de afinidade e intercambio iônico) e ativados com uroquinase, partindo de uma concentração comum e sua cinética avaliada segundo as coordenadas de Lineweaver-Burk. Para isto se utilizou a cinética enzimática de Michaelis-Menten, amplamente usada no estudo de enzimas. Todos os plasminogênios mostraram uma pureza superior a 95 % e uma banda de 92 kDa na eletroforese, ao comparar com o padrão de peso molecular utilizado. As plasminas do equino e canino mostraram a mesma KM pelo substrato cromogênico (0,438 mM), sendo esta a de maior afinidade neste estudo e a humana a de menor afinidade (5,3 mM). Também foram determinadas a constante catalítica e a velocidade de conversão do substrato cromogênico a produto. Os terminais-N dos plasminogênios das oito espécies foram determinados, e se encontraram diferenças entre o humano e os animais, da mesma forma entre alguns animais. Somente o suíno e o ovino não mostraram diferença alguma em seu terminal-N (DPPDDY). Demonstrou-se a unificação do método de purificação de os plasminogênios para qualquer espécie, as diferenças cinéticas das oito plasminas estudadas e as similitudes e diferencias na sequencia dos terminais-N das oito espécies., El artículo determina los parámetros cinéticos de las plasminas de ocho especies de mamíferos y sus terminales-N. Los ocho plasminógenos fueron purificados por los mismos métodos (cromatografías de afinidad e intercambio iónico), y activados con urocinasa, partiendo de una concentración común y su cinética valorada según las coordenadas de Lineweaver- Burk. Para esto se utilizó la cinética enzimática de Michaelis Menten, ampliamente usada en el estudio de enzimas. Todos los plasminógenos mostraron una pureza superior al 95 % y una banda de 92 kDa en la electroforesis, al comparar con el estándar de peso molecular utilizado. Las plasminas del equino y canino mostraron la misma KM por el sustrato cromogénico (0,438 mM), siendo esta la de mayor afinidad en este estudio y la humana la de menor afinidad (5,3 mM). También fueron determinadas la constante catalítica y la velocidad de conversión del sustrato cromogénico a producto. Los terminales-N de los plasminógenos de las ocho especies fueron determinados, y se encontraron diferencias entre el humano y los animales; así mismo entre algunos animales. Solo el porcino y el ovino no mostraron diferencia alguna en su terminal-N (DPPDDY). Se demostró la unificación del método de purificación de los plasminógenos para cualquier especie, las diferencias cinéticas de las ocho plasminas estudiadas, y las similitudes y diferencias en la secuencia de los terminales-N de las ocho especies.

The paper determines the kinetic parameters of plasmins from eight species of mammals and their N-terminals. They were purified by the same methods (affinity chromatography and ion exchange) and activated with urokinase, starting from a common concentration and its kinetics judged according to Lineweaver-Burk coordinates. For such purpose, MichaelisMenten enzyme kinetics, which is widely used in the study of enzymes, was implemented. When compared with the molecular weight standard used, all plasminogen showed a purity exceeding 95% and a 92 kDa band on electrophoresis. Equine and canine plasmins showed the same KM due to the chromogenic substrate (0.438 mM), this being the one with the highest affinity in this study, and the human being the one with the lower affinity (5.3 mM). The catalytic constant and the conversion rate of the chromogenic substrate to product were also determined. The N-terminals of the plasminogens of the eight species were determined, and differences were found between humans and animals, as well as between some animals. Only pigs and sheep showed no differences in their N-terminal (DPPDDY). The unification of the method for purification of plasminogens for any species was demonstrated, as well as the kinetic differences of the eight plasmins studied and the similarities and differences in the sequence the N-terminals of the eight species., El artigo determina os parâmetros cinéticos das plasminas de oito espécies de mamíferos e seus terminais-N. Estes foram purificados pelos mesmos métodos (cromatografias de afinidade e intercambio iônico) e ativados com uroquinase, partindo de uma concentração comum e sua cinética avaliada segundo as coordenadas de Lineweaver-Burk. Para isto se utilizou a cinética enzimática de Michaelis-Menten, amplamente usada no estudo de enzimas. Todos os plasminogênios mostraram uma pureza superior a 95 % e uma banda de 92 kDa na eletroforese, ao comparar com o padrão de peso molecular utilizado. As plasminas do equino e canino mostraram a mesma KM pelo substrato cromogênico (0,438 mM), sendo esta a de maior afinidade neste estudo e a humana a de menor afinidade (5,3 mM). Também foram determinadas a constante catalítica e a velocidade de conversão do substrato cromogênico a produto. Os terminais-N dos plasminogênios das oito espécies foram determinados, e se encontraram diferenças entre o humano e os animais, da mesma forma entre alguns animais. Somente o suíno e o ovino não mostraram diferença alguma em seu terminal-N (DPPDDY). Demonstrou-se a unificação do método de purificação de os plasminogênios para qualquer espécie, as diferenças cinéticas das oito plasminas estudadas e as similitudes e diferencias na sequencia dos terminais-N das oito espécies., El artículo determina los parámetros cinéticos de las plasminas de ocho especies de mamíferos y sus terminales-N. Los ocho plasminógenos fueron purificados por los mismos métodos (cromatografías de afinidad e intercambio iónico), y activados con urocinasa, partiendo de una concentración común y su cinética valorada según las coordenadas de Lineweaver- Burk. Para esto se utilizó la cinética enzimática de Michaelis Menten, ampliamente usada en el estudio de enzimas. Todos los plasminógenos mostraron una pureza superior al 95 % y una banda de 92 kDa en la electroforesis, al comparar con el estándar de peso molecular utilizado. Las plasminas del equino y canino mostraron la misma KM por el sustrato cromogénico (0,438 mM), siendo esta la de mayor afinidad en este estudio y la humana la de menor afinidad (5,3 mM). También fueron determinadas la constante catalítica y la velocidad de conversión del sustrato cromogénico a producto. Los terminales-N de los plasminógenos de las ocho especies fueron determinados, y se encontraron diferencias entre el humano y los animales; así mismo entre algunos animales. Solo el porcino y el ovino no mostraron diferencia alguna en su terminal-N (DPPDDY). Se demostró la unificación del método de purificación de los plasminógenos para cualquier especie, las diferencias cinéticas de las ocho plasminas estudiadas, y las similitudes y diferencias en la secuencia de los terminales-N de las ocho especies.

Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus (GAS), a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator (uPA) contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase (Ska) was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA−/− mice. The observed decrease in virulence in uPA−/−mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.

Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus (GAS), a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator (uPA) contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase (Ska) was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA−/− mice. The observed decrease in virulence in uPA−/−mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.

Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus (GAS), a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator (uPA) contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase (Ska) was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA−/− mice. The observed decrease in virulence in uPA−/−mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.

Staphylococcus aureus peritonitis is a serious complication of peritoneal dialysis (PD). Since reports of the course and treatment of S. aureus peritonitis have generally been limited to small, single-center studies, the aim of the current investigation was to examine the frequency, predictors, treatment, and clinical outcomes of this condition in all 4675 patients receiving PD in Australia between 1 October 2003 and 31 December 2006. 3594 episodes of peritonitis occurred in 1984 patients and 503 (14%) episodes of S. aureus peritonitis occurred in 355 (8%) individuals. 273 (77%) patients experienced 1 episode of S. aureus peritonitis, 52 (15%) experienced 2 episodes, 19 (5%) experienced 3 episodes, and 11 (3%) experienced 4 or more episodes. The predominant antibiotics used as initial empiric therapy were vancomycin (61%) and cephazolin(31%). Once S. aureus was isolated and identified, the prescription of vancomycin did not appreciably change for methicillin-sensitive S. aureus (MSSA) peritonitis (59%) and increased for methicillin-resistant S. aureus (MRSA) peritonitis (84%). S. aureus peritonitis was associated with a higher rate of relapse than non-S. aureus peritonitis (20% vs 13%, p < 0.001) but comparable rates of hospitalization (67% vs 70%, p = 0.2), catheter removal (23% vs 21%, p = 0.4), hemodialysis transfer (18% vs 18%, p = 0.6), and death (2.2% vs 2.3%, p = 0.9). MRSA peritonitis was independently predictive of an increased risk of permanent hemodialysis transfer [odds ratio (OR) 2.11, 95% confidence interval (CI) 1.17 - 3.82] and tended to be associated with an increased risk of hospitalization (OR 2.00, 95% CI 0.96 - 4.19). The initial empiric antibiotic choice between vancomycin and cephazolin was not significantly associated with clinical outcomes, but serious adverse outcomes were more likely if vancomycin was not used for subsequent treatment of MRSA peritonitis. In conclusion, S. aureus peritonitis is a serious complication of PD, involves a smal

The plasminogen/plasmin system has the potential to affect the outcome of inflammatory diseases by regulating accumulation of fibrin and other matrix proteins. In human and experimental crescentic glomerulonephritis (GN), fibrin is an important mediator of glomerular injury and renal impairment. Glomerular deposition of matrix proteins is a feature of progressive disease. To study the role of plasminogen and plasminogen activators in the development of inflammatory glomerular injury, GN was induced in mice in which the genes for these proteins had been disrupted by homologous recombination. Deficiency of plasminogen or combined deficiency of tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA) was associated with severe functional and histological exacerbation of glomerular injury. Deficiency of tPA, the predominant plasminogen activator expressed in glomeruli, also exacerbated disease. uPA deficiency reduced glomerular macrophage infiltration and did not significantly exacerbate disease. uPA receptor deficiency did not effect the expression of GN. These studies demonstrate that plasminogen plays an important role in protecting the glomerulus from acute inflammatory injury and that tPA is the major protective plasminogen activator.

The capacity of macrophages to influence directly and indirectly fibrinolytic processes in atherosclerosis was studied using macrophages isolated from atherosclerotic plaques of patients undergoing surgical repair of distal aortic and femoral arteries. These cells were characterized by their morphology, adherence, esterase positivity, and expression of CD14 antigen. Production of plasminogen activator inhibitor type-1 (PAI-1) by plaque macrophages (6.7 +/- 2.7 ng/105 cells/24 hours [mean +/- SEM]) was significantly greater than PAI-1 production by blood monocytes isolated simultaneously from the same patients (1.8 +/- 1.5 ng/105 cells/24 hours). Production of tissue type plasminogen activator and urokinase type plasminogen activator by plaque macrophages was not augmented compared to blood monocytes. Conditioned medium from cultured plaque macrophages significantly increased production of PAI-1 by endothelial cells (85 +/- 11% above basal) and vascular smooth muscle cells (25 +/- 10%) in vitro. This response was significantly greater than the response to monocyte-conditioned medium (endothelial cells 38 +/- 11%, vascular smooth muscle cells 2.5 +/- 2.0%). Stimulation of endothelial cell PAI-1 production by macrophage- conditioned medium was partially inhibitable by a monoclonal antibody to transforming growth factor-beta. Tissue type plasminogen activator production by endothelial cells and vascular smooth muscle cells was not affected by plaque macrophage- or monocyte-conditioned medium. Urokinase type plasminogen activator production by endothelial cells and vascular smooth muscle cells was undetectable in control medium and was augmented to similar levels in response to plaque macrophage- and monocyte-conditioned media. These results demonstrate up-regulation of PAI-1 production by macrophages in atheromatous plaques and the capacity of soluble products from plaque macrophages to up- regulate PAI-1 production by endothelial cells and vascular smooth muscle cells

Staphylococcus aureus peritonitis is a serious complication of peritoneal dialysis (PD). Since reports of the course and treatment of S. aureus peritonitis have generally been limited to small, single-center studies, the aim of the current investigation was to examine the frequency, predictors, treatment, and clinical outcomes of this condition in all 4675 patients receiving PD in Australia between 1 October 2003 and 31 December 2006. 3594 episodes of peritonitis occurred in 1984 patients and 503 (14%) episodes of S. aureus peritonitis occurred in 355 (8%) individuals. 273 (77%) patients experienced 1 episode of S. aureus peritonitis, 52 (15%) experienced 2 episodes, 19 (5%) experienced 3 episodes, and 11 (3%) experienced 4 or more episodes. The predominant antibiotics used as initial empiric therapy were vancomycin (61%) and cephazolin(31%). Once S. aureus was isolated and identified, the prescription of vancomycin did not appreciably change for methicillin-sensitive S. aureus (MSSA) peritonitis (59%) and increased for methicillin-resistant S. aureus (MRSA) peritonitis (84%). S. aureus peritonitis was associated with a higher rate of relapse than non-S. aureus peritonitis (20% vs 13%, p < 0.001) but comparable rates of hospitalization (67% vs 70%, p = 0.2), catheter removal (23% vs 21%, p = 0.4), hemodialysis transfer (18% vs 18%, p = 0.6), and death (2.2% vs 2.3%, p = 0.9). MRSA peritonitis was independently predictive of an increased risk of permanent hemodialysis transfer [odds ratio (OR) 2.11, 95% confidence interval (CI) 1.17 - 3.82] and tended to be associated with an increased risk of hospitalization (OR 2.00, 95% CI 0.96 - 4.19). The initial empiric antibiotic choice between vancomycin and cephazolin was not significantly associated with clinical outcomes, but serious adverse outcomes were more likely if vancomycin was not used for subsequent treatment of MRSA peritonitis. In conclusion, S. aureus peritonitis is a serious complication of PD, involves a smal

The plasminogen/plasmin system has the potential to affect the outcome of inflammatory diseases by regulating accumulation of fibrin and other matrix proteins. In human and experimental crescentic glomerulonephritis (GN), fibrin is an important mediator of glomerular injury and renal impairment. Glomerular deposition of matrix proteins is a feature of progressive disease. To study the role of plasminogen and plasminogen activators in the development of inflammatory glomerular injury, GN was induced in mice in which the genes for these proteins had been disrupted by homologous recombination. Deficiency of plasminogen or combined deficiency of tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA) was associated with severe functional and histological exacerbation of glomerular injury. Deficiency of tPA, the predominant plasminogen activator expressed in glomeruli, also exacerbated disease. uPA deficiency reduced glomerular macrophage infiltration and did not significantly exacerbate disease. uPA receptor deficiency did not effect the expression of GN. These studies demonstrate that plasminogen plays an important role in protecting the glomerulus from acute inflammatory injury and that tPA is the major protective plasminogen activator.

The capacity of macrophages to influence directly and indirectly fibrinolytic processes in atherosclerosis was studied using macrophages isolated from atherosclerotic plaques of patients undergoing surgical repair of distal aortic and femoral arteries. These cells were characterized by their morphology, adherence, esterase positivity, and expression of CD14 antigen. Production of plasminogen activator inhibitor type-1 (PAI-1) by plaque macrophages (6.7 +/- 2.7 ng/105 cells/24 hours [mean +/- SEM]) was significantly greater than PAI-1 production by blood monocytes isolated simultaneously from the same patients (1.8 +/- 1.5 ng/105 cells/24 hours). Production of tissue type plasminogen activator and urokinase type plasminogen activator by plaque macrophages was not augmented compared to blood monocytes. Conditioned medium from cultured plaque macrophages significantly increased production of PAI-1 by endothelial cells (85 +/- 11% above basal) and vascular smooth muscle cells (25 +/- 10%) in vitro. This response was significantly greater than the response to monocyte-conditioned medium (endothelial cells 38 +/- 11%, vascular smooth muscle cells 2.5 +/- 2.0%). Stimulation of endothelial cell PAI-1 production by macrophage- conditioned medium was partially inhibitable by a monoclonal antibody to transforming growth factor-beta. Tissue type plasminogen activator production by endothelial cells and vascular smooth muscle cells was not affected by plaque macrophage- or monocyte-conditioned medium. Urokinase type plasminogen activator production by endothelial cells and vascular smooth muscle cells was undetectable in control medium and was augmented to similar levels in response to plaque macrophage- and monocyte-conditioned media. These results demonstrate up-regulation of PAI-1 production by macrophages in atheromatous plaques and the capacity of soluble products from plaque macrophages to up- regulate PAI-1 production by endothelial cells and vascular smooth muscle cells

Tumor-specific delivery of ligand-directed prodrugs can increase the therapeutic window of chemotherapeutics by maintaining efficacy whilst decreasing toxic side effects. We have previously described a series of synthetic N-alkylated isatin cytotoxins that destabilize microtubules and induce apoptosis with 10-fold greater potency than conventional anti-mitotics in vitro. Here, we report the characterization, in vitro cytotoxicity and in vivo efficacy of a lead compound, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (N-AI) conjugated via an esterase-labile linker (N-AIE) to two proven targeting ligands, transferrin (Tf) and plasminogen activator inhibitor type 2 (PAI-2/serpinB2). N-AI was released from N-AIE and the targeting ligands Tf/PAI-2 in an esterase-dependent manner at 37 °C and both Tf- and PAI-2-N-AIE conjugates were stable at physiological pH. Human cancer cell lines which vary in their expression levels of Tf receptor (TfR/CD71) and PAI-2 target, receptor bound urokinase (uPA) selectively internalized the conjugates. Tf-N-AIE was up to 24 times more active than the free drug and showed clear selectivity patterns based on TfR levels. PAI-2-N-AIE showed equivalent activity compared to the parent drug and strong selectivity patterns for uPA levels. In preliminary in vivo experiments, the PAI-2- and Tf-N-AIE conjugates were efficacious at 1/20th and 1/10th of the dose of the free N-AI, respectively, in a metastatic, orthotopic human breast tumor xenograft mouse model. Thus, this strategy specifically delivers and concentrates a novel class of isatin-based, tubulin destabilizing agents to tumors in vivo and warrants further detailed preclinical investigation.

Tumor-specific delivery of ligand-directed prodrugs can increase the therapeutic window of chemotherapeutics by maintaining efficacy whilst decreasing toxic side effects. We have previously described a series of synthetic N-alkylated isatin cytotoxins that destabilize microtubules and induce apoptosis with 10-fold greater potency than conventional anti-mitotics in vitro. Here, we report the characterization, in vitro cytotoxicity and in vivo efficacy of a lead compound, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (N-AI) conjugated via an esterase-labile linker (N-AIE) to two proven targeting ligands, transferrin (Tf) and plasminogen activator inhibitor type 2 (PAI-2/serpinB2). N-AI was released from N-AIE and the targeting ligands Tf/PAI-2 in an esterase-dependent manner at 37 °C and both Tf- and PAI-2-N-AIE conjugates were stable at physiological pH. Human cancer cell lines which vary in their expression levels of Tf receptor (TfR/CD71) and PAI-2 target, receptor bound urokinase (uPA) selectively internalized the conjugates. Tf-N-AIE was up to 24 times more active than the free drug and showed clear selectivity patterns based on TfR levels. PAI-2-N-AIE showed equivalent activity compared to the parent drug and strong selectivity patterns for uPA levels. In preliminary in vivo experiments, the PAI-2- and Tf-N-AIE conjugates were efficacious at 1/20th and 1/10th of the dose of the free N-AI, respectively, in a metastatic, orthotopic human breast tumor xenograft mouse model. Thus, this strategy specifically delivers and concentrates a novel class of isatin-based, tubulin destabilizing agents to tumors in vivo and warrants further detailed preclinical investigation.

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-tumour/metastasis agents and several approaches have been explored for this purpose, including the use of small molecule antagonists. Two highly potent non-peptidic antagonists 1 and 2 (IC50 1 = 0.8 nM, IC50 2 = 33 nM) from the patent literature were reportedly identified using competition assays employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their potencies in physiologically relevant cell-based competition assays employing uPAR's native binding partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employed HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations of 2 (10-100 μM) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antagonist effects at 100 μM. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor inhibitors of cell surface-bound HMW-uPA activity (IC50 >100 μM for 1 and 2). The report highlights the dangers of identifying false-positive lead uPAR antagonists from competition assays employing labelled competing ligands other than the native HMW-uPA. © 2011 Elsevier Ltd. All rights reserved.

Up-regulation of the urokinase plasminogen activation (uPA) system leads to increased cancer invasion and metastasis. Plasminogen activator inhibitors type-1 (PAI-1/SERPINE1) and type-2 (PAI-2/SERPINB2) have similar uPA inhibitory properties yet PAI-1 promotes cell invasion by modulating cell adhesion and migration. High tumour PAI-2 levels are associated with improved prognoses. Herein we show that PAI-2 is capable of inhibiting uPA in the presence of PAI-1, particularly on adherent cells in the presence of vitronectin. This suggests that elevated levels of PAI-2 in the tumour microenvironment could outcompete PAI-1 for uPA inhibition through its inhibitory serpin function. However, PAI-1 modulated cell adhesion in a largely uPA-independent manner consequently PAI-2 could not counteract the effects of PAI-1 on adhesion/migration. Thus studies aimed at further characterising the interplay between PAI-1 and PAI-2 on uPA-dependent pro-invasive processes are warranted.

Up-regulation of the urokinase plasminogen activation (uPA) system leads to increased cancer invasion and metastasis. Plasminogen activator inhibitors type-1 (PAI-1/SERPINE1) and type-2 (PAI-2/SERPINB2) have similar uPA inhibitory properties yet PAI-1 promotes cell invasion by modulating cell adhesion and migration. High tumour PAI-2 levels are associated with improved prognoses. Herein we show that PAI-2 is capable of inhibiting uPA in the presence of PAI-1, particularly on adherent cells in the presence of vitronectin. This suggests that elevated levels of PAI-2 in the tumour microenvironment could outcompete PAI-1 for uPA inhibition through its inhibitory serpin function. However, PAI-1 modulated cell adhesion in a largely uPA-independent manner consequently PAI-2 could not counteract the effects of PAI-1 on adhesion/migration. Thus studies aimed at further characterising the interplay between PAI-1 and PAI-2 on uPA-dependent pro-invasive processes are warranted.

A known side-activity of the oral potassium-sparing diuretic drug amiloride is inhibition of the enzyme urokinase-type plasminogen activator (uPA, Ki = 7 mu M), a promising anticancer target. Several studies have demonstrated significant antitumor/metastasis properties for amiloride in animal cancer models and it would appear that these arise, at least in part, through inhibition of uPA. Selective optimization of amiloride’s structure for more potent inhibition of uPA and loss of diuretic effects would thus appear as an attractive strategy towards novel anticancer agents. The following report is a preliminary structure-activity exploration of amiloride analogs as inhibitors of uPA. A key finding was that the well-studied 5-substituted analogs ethylisopropyl amiloride (EIPA) and hexamethylene amiloride (HMA) are approximately 2-fold more potent than amiloride as uPA inhibitors.

from the 41st Annual Scientific Meeting of the Australian and New Zealand Society of Nuclear Medicine, 14-18 July 2011, Darwin, Australia

A known side-activity of the oral potassium-sparing diuretic drug amiloride is inhibition of the enzyme urokinase-type plasminogen activator (uPA, Ki = 7 mu M), a promising anticancer target. Several studies have demonstrated significant antitumor/metastasis properties for amiloride in animal cancer models and it would appear that these arise, at least in part, through inhibition of uPA. Selective optimization of amiloride’s structure for more potent inhibition of uPA and loss of diuretic effects would thus appear as an attractive strategy towards novel anticancer agents. The following report is a preliminary structure-activity exploration of amiloride analogs as inhibitors of uPA. A key finding was that the well-studied 5-substituted analogs ethylisopropyl amiloride (EIPA) and hexamethylene amiloride (HMA) are approximately 2-fold more potent than amiloride as uPA inhibitors.

Urokinase (uPA) is a 411 residues serine protease originally identified for its ability to activate plasminogen and generate plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme. Later, this protease has been shown to possess also a clear-cut ability to stimulate cell migration and survival in a catalytic-independent manner. This activity turned out to be exerted through the growth factor-like domain (GFD-like, residues 1-49) of the protease binding to a GPI-anchored membrane receptor (uPAR), in complex with transmembrane receptors such as integrins, the epidermal growth factor and the formyl-peptide receptors. Direct binding of uPA to integrins through its kringle (residues 50-131) and connecting peptide (residues 132-158) regions results in enhanced migration. The dual function of uPA in promoting migration while reducing the physical resistance of extracellular matrix underlies its crucial role in the invasion of malignant tumours. Consolidated evidence emerging from animal models and clinical studies shows that the overexpression of uPA is a causal determinant to tumour metastasis and is associated to a poor prognosis. Therefore, pinpointing the molecular interactions and identifying novel agents to interfere with the diverse activities of uPA is a goal of basic and applied research. In this review, we discuss the general theme of cell migration and invasion. A description of the uPA structure-function relationship and the functional effects of isolated domains is presented. Current information on molecular agonistic as well as antagonistic compounds, including the compounds which have reached clinical trials, is provided. © 2011 Bentham Science Publishers.

Acute myeloid leukemia (AM L) is a hematological malignancy characterized by the rapid expansion of immature clonal myeloid cells, which leads to the failure of normal hematopoiesis. The remission rate in AML patients, following induction therapy, is 50 to 70% but most patients who undergo complete remission, recur later on with a poor prognosis. Thus leukemic re lapse in AM L remains a major therapeutic problem with the need of developing novel, tumor specific AML therapies. We aim to selectively target AM L cells using a recombinant anthrax lethal toxin (LeTx) and a urokinase-activated recombinant anthrax toxin (PrAgU2/LF). LeTx is a binary toxin consisting of 2 proteins, protective antigen (PrAg), and lethal factor (LF). PrAg binds cells through the anthrax toxin receptors (ANTXRs) and is cleaved by cell surface furin proteases leading to the release of a 20 kOa fragment and the generation of an activated 63 kOa PrAg protein. Active PrAg forms heptamers, binds 3 molecu les of LF and undergoes receptor-mediated endocytosis followed by release of LF into the cytosol. LF is a zinc metalloprotease that cleaves and inactivates mitogenactivated protein kinase (MAPK) kinases leading to the inactivation of the MAPK pathway. A modified, urokinase-activated version of anthrax lethal toxin (PrAgU2/LF) in which the furin cleavage sequence of PrAg is substituted with a sequence cleaved by the urokinase plasminogen activator (uPA/uPAR) protease system was generated. The MAPK pathway and the uPA/uPAR system have been shown to be active in a number of tumors, including AML, rendering AML cells potential targets for both LeTx (PrAg/LF) and the dual-specific, urokinase-activated PrAgU2/LF. In this study, we tested the sensitivity of a panel of AML cell lines to LeTx (PrAg/LF), PrAgU2/LF, PrAg/FP59 and PrAgU2/FP59 using a proliferation inhibition (cytotoxicity) assay. In addition, we targeted the PI3K1AKT pathway using the PI3K inhibitor LY294002 and the Ras-Raf-MEK1 /2-ERK1/2 path

A known side-activity of the oral potassium-sparing diuretic drug amiloride is inhibition of the enzyme urokinase-type plasminogen activator (uPA, Ki = 7 mu M), a promising anticancer target. Several studies have demonstrated significant antitumor/metastasis properties for amiloride in animal cancer models and it would appear that these arise, at least in part, through inhibition of uPA. Selective optimization of amiloride’s structure for more potent inhibition of uPA and loss of diuretic effects would thus appear as an attractive strategy towards novel anticancer agents. The following report is a preliminary structure-activity exploration of amiloride analogs as inhibitors of uPA. A key finding was that the well-studied 5-substituted analogs ethylisopropyl amiloride (EIPA) and hexamethylene amiloride (HMA) are approximately 2-fold more potent than amiloride as uPA inhibitors.

A known side-activity of the oral potassium-sparing diuretic drug amiloride is inhibition of the enzyme urokinase-type plasminogen activator (uPA, Ki = 7 mu M), a promising anticancer target. Several studies have demonstrated significant antitumor/metastasis properties for amiloride in animal cancer models and it would appear that these arise, at least in part, through inhibition of uPA. Selective optimization of amiloride’s structure for more potent inhibition of uPA and loss of diuretic effects would thus appear as an attractive strategy towards novel anticancer agents. The following report is a preliminary structure-activity exploration of amiloride analogs as inhibitors of uPA. A key finding was that the well-studied 5-substituted analogs ethylisopropyl amiloride (EIPA) and hexamethylene amiloride (HMA) are approximately 2-fold more potent than amiloride as uPA inhibitors.

Up-regulation of the urokinase plasminogen activation (uPA) system leads to increased cancer invasion and metastasis. Plasminogen activator inhibitors type-1 (PAI-1/SERPINE1) and type-2 (PAI-2/SERPINB2) have similar uPA inhibitory properties yet PAI-1 promotes cell invasion by modulating cell adhesion and migration. High tumour PAI-2 levels are associated with improved prognoses. Herein we show that PAI-2 is capable of inhibiting uPA in the presence of PAI-1, particularly on adherent cells in the presence of vitronectin. This suggests that elevated levels of PAI-2 in the tumour microenvironment could outcompete PAI-1 for uPA inhibition through its inhibitory serpin function. However, PAI-1 modulated cell adhesion in a largely uPA-independent manner consequently PAI-2 could not counteract the effects of PAI-1 on adhesion/migration. Thus studies aimed at further characterising the interplay between PAI-1 and PAI-2 on uPA-dependent pro-invasive processes are warranted.

from the 41st Annual Scientific Meeting of the Australian and New Zealand Society of Nuclear Medicine, 14-18 July 2011, Darwin, Australia

Up-regulation of the urokinase plasminogen activation (uPA) system leads to increased cancer invasion and metastasis. Plasminogen activator inhibitors type-1 (PAI-1/SERPINE1) and type-2 (PAI-2/SERPINB2) have similar uPA inhibitory properties yet PAI-1 promotes cell invasion by modulating cell adhesion and migration. High tumour PAI-2 levels are associated with improved prognoses. Herein we show that PAI-2 is capable of inhibiting uPA in the presence of PAI-1, particularly on adherent cells in the presence of vitronectin. This suggests that elevated levels of PAI-2 in the tumour microenvironment could outcompete PAI-1 for uPA inhibition through its inhibitory serpin function. However, PAI-1 modulated cell adhesion in a largely uPA-independent manner consequently PAI-2 could not counteract the effects of PAI-1 on adhesion/migration. Thus studies aimed at further characterising the interplay between PAI-1 and PAI-2 on uPA-dependent pro-invasive processes are warranted.