Synergistic cytotoxicity of a prostate cancer-specific immunotoxin in combination with the BH3 mimetic ABT-737.

In many tumors, including prostate cancer, anti-apoptotic members of the Bcl-2 family are overexpressed and cause cell death resistance, which is a typical hallmark of cancer. Different therapeutic approaches, therefore, aim to restore the death mechanisms for enhanced apoptosis. Our recombinant immunotoxin D7(VL-VH)-PE40 is composed of the scFv D7(VL-VH) against the prostate-specific membrane antigen (PSMA) on the surface of prostate cancer cells and of the cytotoxic domain of the bacterial toxin Pseudomonas Exotoxin A (PE40). Since Pseudomonas Exotoxin A-based immunotoxins are known to preferentially inhibit the expression of the anti-apoptotic protein Mcl-1, the rationale was to test our immunotoxin in combination with the BH3 mimetic ABT-737, which specifically inhibits Bcl-2, Bcl-xl, and Bcl-w for enhanced induction of apoptosis in prostate cancer cells. The immunotoxin showed high and specific binding and cytotoxicity against PSMA expressing prostate cancer cells marked by a direct inhibition of Mcl-1. The combination of the immunotoxin with a subtoxic concentration of ABT-737 caused additive or even synergistic effects, which were based on an enhanced apoptosis induction as detected by poly(ADP-ribose) polymerase (PARP) and Caspase-3 cleavage in Western blot. Our study shows that the combination therapy of immunotoxin plus ABT-737 is a promising approach for the future treatment of advanced prostate cancer to improve therapeutic efficacy and to reduce adverse side effects.

Contemporary follow-up imaging after endovascular repair of lower extremity atherosclerotic lesions.

Atherosclerotic disease is currently one of the most important problems of modern medicine because it is a leading cause of increased morbidity, morbidity and mortality, and disability in the Western World. Atherosclerosis of the lower limbs (peripheral arterial disease - PAD) significantly affects the quality of life and in a considerable proportion of patients is a cause of disability. Radical treatment of PAD, both surgical and endovascular, aims at revascularisation of ischaemic tissues distal to obstructed arteries. Surveillance imaging is an important part of patient management after endovascular repair of PAD. Apart from availability and contraindications, challenges of imaging include calcifications, flow dynamics, and stent-related artefacts. The aim of this paper was to review the current literature on imaging methods for follow-up after endovascular repair of atherosclerotic lesions, with special attention paid to novel techniques. As a non-invasive modality, ultrasound is still the first-line examination, but computed tomography angiography remains a current state-of-the art technique for follow-up. However, since current imaging recommendations seem not to adhere to contemporary imaging possibilities, more attention should be paid to recent improvements in magnetic resonance angiography technology.

Estimation of the use of fibrin and collagen membranes as carriers for platelet-derived growth factor-BB (PDGF-BB) in the presence of amoxicillin.

The effect of homogeneous fibrin (Fb), collagen (Coll) and composite fibrin-heparin (Fb-Hp), fibrin-collagen (Fb-Coll) membranes on in vitro release of platelet-derived growth factor (PDGF-BB) was evaluated in the presence or absence of amoxicillin using of the ELISA immunoassay test. Amoxicillin concentration was determined spectrophotometrically at 272 nm. The process of the PDGF-BB growth factor and amoxicillin release from the studied membranes was of a two-phase nature in the majority of the systems analysed. The PDGF-BB was released in the highest amount from the Coll membrane (M7) without the presence of amoxicillin--546.2 ± 7.47 pg, t0.5 = 0.88 h and 202.5 ± 6.83 pg, t0.5 = 26.65 h during the first phase and second phase, respectively. The lowest PDGF-BB release was observed from composite M4 (Fb-Hp) membrane--5.88 ± 0.81 pg, t0.5 = 1.69 h; and 110.2 ± 6.48 pg, t0.5 = 855.6 h during first and second phase respectively. An optimal release of amoxicillin was observed in the case of the composite M6 (Fb-Coll) membrane--only in the second phase: 64.2 ± 7.8 µg, t0.5 = 83.5 h. The lowest and delayed amoxicillin release was achieved for M4 membrane (approx. 17.1 ± 1.12 µg, t0.5 = 46.5 h). The results of the PDGF-BB release and amoxicillin from membranes indicated a correlation between the level of release and composition of the film. Our results suggested that fibrin and collagen membranes may be beneficial to enhance periodontal bone regeneration.

Fibrinogen, bFGF and VEGF levels during antibiotic therapy in gynecologic cancer: a preliminary report.

The role of angiogenesis in the development of neoplasia has been identified and characterized. However, antiangiogenic therapeutic intervention still requires more evidence to become recognized and successful. The aim of this study was to evaluate levels of selected proangiogenic factors, such as fibrinogen, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in serum of patients with the gynecologic cancer on the first, third and sixth day of antibiotic therapy, routinely administered as a perioperative treatment. In addition, serum concentrations of gamma-gamma dimers and alpha-polymers of cross-linked fibrin structure and the degree of bFGF binding with the fibrin network were investigated. Immunohistochemistry staining of the excised tumor tissue was also performed. We observed higher levels of bFGF, VEGF, as well as fibrinogen in patients with gynecologic malignancy, as compared to healthy women. In cancer patients, the concentration of alpha-polymers and gamma-gamma dimers of fibrin network increased. Further only gamma-gamma dimers fraction of fibrin was found to bind to bFGF. Immunohistochemical analysis indicated the presence of bFGF in an excised tumor tissue. In conclusion, the decrease of proangiogenic bFGF and fibrinogen levels in a clinical trial of gynecologic patients may confirm anti-angiogenic properties of selected antibiotic therapy.

Visualization of vascular inflammation in the atherosclerotic mouse by ultrasmall superparamagnetic iron oxide vascular cell adhesion molecule-1-specific nanoparticles.

OBJECTIVE: Noninvasive imaging of atherosclerosis remains challenging in clinical applications. Here, we applied noninvasive molecular imaging to detect vascular cell adhesion molecule-1 in early and advanced atherosclerotic lesions of apolipoprotein E-deficient mice. METHODS AND RESULTS: Ultrasmall superparamagnetic iron oxide particles functionalized with (P03011) or without (P3007) vascular cell adhesion molecule-1-binding peptide were visualized by ultra high-field (17.6 T) magnetic resonance. Injection of P03011 resulted in a marked signal loss in the aortic root of apolipoprotein E-deficient mice fed a Western diet for 8 and 26 weeks in vivo and ex vivo, compared with preinjection measurements, P3007-injected mice, and P03011- or P3007-injected age-matched C57BL/6 controls. Histological analyses revealed iron accumulations in the intima, in colocalization with vascular cell adhesion molecule-1-expressing macrophages and endothelial cells. Coherent anti-Stokes Raman scattering microscopy demonstrated iron signals in the intima and media of the aortic root in the P03011-injected but not untreated apolipoprotein E-deficient mice, localized to macrophages, luminal endothelial-like cells, and medial regions containing smooth muscle cells. Electron microscopy confirmed iron particles enclosed in endothelial cells and in the vicinity of smooth muscle cells. CONCLUSIONS: Using a combination of innovative imaging modalities, in this study, we demonstrate the feasibility of applying P03011 as a contrast agent for imaging of atherosclerosis.

Effect of phosphorohydrazone derivatives of chromone on fibrin polymerization in the presence of bFGF.

The effect of ten phosphorohydrazone derivatives of chromone on the fibrin formation process was evaluated in the presence of basic fibroblast growth factor (bFGF), which plays an important role in tumor angiogenesis. The relationship between the chemical properties of the derivatives and the resulting fibrin structure was also examined. The structure of fibrin was analyzed by UV spectrophotometry and the degree of bFGF binding to fibrin was estimated by Western blotting. The measurements were taken at 3 different pH values: 6.64, 7.60 and 8.40. Three of the analyzed phosphorohydrazone derivatives (compounds 2, 7 and 10) demonstrated the most significant influence on reduction of polymerization at the studied pH values.

The role of polyphenols in cardiovascular disease.

Epidemiological studies suggest that diets rich in polyphenols may be associated with reduced incidence of cardiovascular disorders (mainly coronary heart disease and myocardial infarction). However, the mechanisms explaining this correlation have not been fully elucidated. Current evidence suggests that polyphenols, acting at the molecular level, improve endothelial function and inhibit platelet aggregation. In view of their antithrombotic, anti-inflammatory, and anti-aggregative properties, these compounds may play a roles in the prevention and treatment of cardiovascular disease.

Influence of phosphorohydrazone derivatives of benzopyrones on polymerization and viscosity of fibrin.

The synthesis and antitumour and antibacterial activity of coumarin and chromone phosphorohydrazones have been reported. This study describes influence of phosphorohydrazones derivatives of coumarin and chromone on the polymerization and viscosity of fibrin. The fibrin polymerization assay was performed by the Shen and Lorand method and the clot viscosity was measured on the basis of Shen and Lorand and Marchi and coworkers methods. Among the eight compounds tested, one coumarin derivative and two chromone derivatives showed significant activity.

Polymer angiogenic factor carrier. Part I. Chitosan-alginate membrane as carrier PDGF-AB and TGF-beta.

Microcrystalline chitosan (MCCh) characterise by many valuable features, including biocompatibility, antibacterial, fungicidal and high adhesive properties, high water retention value (WRV) factor, potent sorption, high bioactivity, biodegradability, ability to create membranes directly out of water suspensions. This biopolymer due to its biodegradability and safety in application no cytotoxic or immune interaction was assessed as a very effective haemostatic factor. MCCh with such properties form useful initial material for biological bandages; it can be used in surgery as growth factor carrier. Biodegradable mambranes are applied in surgery for guided tissue regeneration, what contributed to make the manufacturing technology of modified membranes out of microcrystalline chitosan. Peformed research was to show how certain angiogenic factors are released--platelet derived angiogenic factor (PDAF-AB) and transforming factor beta (TGF-beta) from doubled-layered alginate-chitosane membrane which was already used in dental surgery. Assessment of angiogenic factors release (PDGF-AB and TGF-beta) from polymer base may become helpful in choosing the right carrier for growth factors.

In Vitro Evaluation of Humanized/De-immunized Anti-PSMA Immunotoxins for the Treatment of Prostate Cancer.

BACKGROUND: We generated humanized/de-immunized immunotoxins targeting the prostate-specific membrane antigen (PSMA) and tested their cytotoxic activity against prostate cancer cells in vitro. MATERIALS AND METHODS: The humanized/de-immunized version of our murine anti-PSMA single-chain antibody fragment (scFv) D7, termed hD7-1(VL-VH), was ligated to the 40-kDa toxin domain of Pseudomonas aeruginosa exotoxin A (PE40), and to the deimmunized 24-kDa toxin domains PE24 or PE24mut. The immunotoxins designated as hD7-1(VL-VH)-PE40, hD7-1(VL-VH)-PE24 and hD7-1(VL-VH)-PE24mut were bacterially expressed and purified by affinity chromatography. Binding and cytotoxicity were examined by flow cytometry and viability assay, respectively. RESULTS: All immunotoxins revealed strong binding to prostate cancer cells expressing PSMA and specific cytotoxicity, with half-maximal inhibitory concentration values in the picomolar range. CONCLUSION: We successfully created powerful anti-PSMA immunotoxins with reduced immunogenicity for further clinical development and application against advanced prostate cancer.

Impact of Methadone on Cisplatin Treatment of Bladder Cancer Cells.

BACKGROUND: Cisplatin-based chemotherapy is the treatment of choice for advanced bladder cancer. Since many tumor cells show inherent or acquired cisplatin resistance, research is needed to improve the therapeutic efficacy. Since the analgesic methadone is discussed as being a sensitizer for chemotherapy, we tested its effects on the cisplatin treatment of bladder cancer cells. MATERIALS AND METHODS: T24 and HT-1376 bladder cancer cells were incubated with cisplatin in combination with methadone. Cytotoxicity was examined using the WST-1 viability assay and induction of apoptosis was analyzed via phase-contrast microscopy, flow cytometry, and western blot analysis. RESULTS: Methadone was shown to enhance the cytotoxic effects of cisplatin on T24 cells based on the induction of apoptosis. In contrast, HT-1376 cells were identified as non-responders to methadone. CONCLUSION: Methadone could act as a chemosensitizer in the future treatment of advanced bladder cancer. Further research is needed to identify the underlying molecular mechanisms.

[Estimation of the correlation between hemostatic parameters and serum lipids in patients with hyperlipidemia type II after simvastatin therapy]. / Ocena korelacji miedzy parametrami hemostazy a lipidogramem surowicy u chorych na hiperlipidemie typu II po leczeniu simwastatyna.

UNLABELLED: The aim of the study was to evaluate the correlation between hemostatic parameters such as: the activity of antithrombin III and factor X, thrombin generation, clot bound thrombin, fibrinogen and serum lipids in patients (pts) with hyperlipidemia (hlp) type II after 2-months treatment with simvastatin in dose 20 mg/day. MATERIAL AND METHODS: The study involved 22 pts with hip with the initial concentrations of total cholesterol (TC) > 230 mg/dL, LDL-cholesterol (LDL-C) > 130 mg/dL, triglicerides (TG) < 400 mg/dL, 22 healthy volunteers as the control group. The hemostatic parameters and serum lipids were estimated before and after active therapy. TC, HDL-C, TG were determined by enzymatic method, LDL-C was calculated by Friedewald'a formula. Thrombin generation and clot bound thrombin levels were estimated by spectrophotometric method with usage of chromogenic substrate S-2238, the activity of factor X and antithrombin III--by amidolytic methods. For correlation we used coefficient of linear correlation. RESULTS: After 2-months therapy with simvastatin in patients with hlp II it was observed significantly decrease of activity of factor X (20.94 +/- 19.04 vs. 9.44 +/- 7.31 mU/mL), thrombin generation (79.62 +/- 36.68 vs. 67.99 +/- 37.69 U/mL), clot bound thrombin (49.73 +/- 21.17 vs. 37.08 +/- 26.10 U/mL) and increase of antithrombin III activity (13.03 +/- 6.11 vs. 20.64 +/- 4.18 U/mL). The mean concentrations of fibrinogen didn't change during statin treatment (336.00 +/- 71.4 vs. 357.26 +/- 98.86 mg/dL). After simvastatin treatment it wasn't observed any significant correlation between the changes of hemostatic parameters and serum lipids. CONCLUSIONS: The short therapy of simvastatin therapy beneficial modifies hemostatic parameters. Lack of linear correlation between lipids changes concentrations and hemostatic parameters during simvastatin treatment suggests hypolipemic independent influence of statin on the coagulation system.

Methadone as a "Tumor Theralgesic" against Cancer.

Methadone has beneficial characteristics as an analgesic against cancer pain, including high bioavailability, multiple receptor affinities, and lack of active metabolites that might induce adverse side effects. However, methadone has an own pharmacological profile that should be considered in the treatment of cancer patients. There is evidence from preclinical studies that methadone could also elicit antitumor activity by downregulating the threshold of apoptosis and to enhance the effects of different chemotherapeutic agents. This confirms the concept of using methadone as a chemosensitizer in the future treatment of cancer. Our article discusses major issues about the role of methadone as a possible "tumor theralgesic," combining tumor therapeutic and analgesic activities.

The effect of hydrazine derivatives of 3-formylchromones on angiogenic basic fibroblast growth factor and fibroblast growth factor receptor-1 in human melanoma cell line WM-115.

The hydrazine derivatives of benzopyrones remain an unexplored group of chemical compounds. This preliminary study investigates the influence of A-5, CH-3 and K-2 derivatives at concentrations of 1, 10, 100 nM and 1 µM on selected biochemical factors of a melanoma cell line WM-115, with regard to their potential angiogenic properties. The studied compounds were found to influence cell proliferation, as well as total protein, bFGF and FGFR1 concentration.

In vitro and in vivo effects of a recombinant anti-PSMA immunotoxin in combination with docetaxel against prostate cancer.

Docetaxel (DOC) is used for the first-line treatment of castration resistant prostate cancer (CPRC). However, the therapeutic effects are limited, only about one half of patients respond to the therapy and severe side effects possibly lead to discontinuation of treatment. Therefore, actual research is focused on the development of new DOC-based combination treatments. In this study we investigated the antitumor effects of a recombinant immunotoxin targeting the prostate specific membrane antigen (PSMA) in combination with DOC in vitro and in vivo. The immunotoxin consists of an anti-PSMA single chain antibody fragment (scFv) as binding and a truncated form of Pseudomonas aeruginosa Exotoxin A (PE40) as toxin domain. The immunotoxin induced apoptosis and specifically reduced the viability of androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cells. A synergistic cytotoxic activity was observed in combination with DOC with IC50 values in the low picomolar or even femtomolar range. Moreover, combination treatment resulted in an enhanced antitumor activity in a C4-2 SCID mouse xenograft model. This highlights the immunotoxin as a promising therapeutic agent for a future DOC-based combination therapy of CPRC.

[The effect of low magnetic field on select parameters of blood coagulation]. / Wplyw pola magnetycznego niskiej czestotliwosci na wybrane parametry ukladu krzepniecia.

UNLABELLED: Low frequency magnetic field causes the biological effects in organisms--in individual systems and organs. OBJECTIVE: The purpose of our study is to analyse the influence of the low magnetic field used in magnetotherapy on prothrombin time, factor Xa activity and level of platelets in experimental animals. MATERIAL AND METHODS: The examination was carried out on rats which were subjected to the activity of low magnetic field. The examination of prothrombin time in Quick's method (the international normalized ratio--INR was counted), activity of factor Xa by spectrophotometric method (lambda = 405 nm) were performed. The level of platelets before and after 14, 28 days of the exposition of magnetic field and after 21 days of exposure to magnetic field was assessed. RESULTS: The extension of prothrombin time (a decrease of INR), decrease activity of factor Xa and decrease platelets level was observed in experimental animals after the exposure to low magnetic field (p < 0.05 for all examined parameters). CONCLUSION: Low magnetic field used in magnetotherapy causes the changes in blood coagulation of experimental animals.

[The comparison of simvastatin and atorvastatin effects on hemostatic parameters in patients with hyperlipidemia type II]. / Porównanie wplywu simwastatyny i atorwastatyny na wybrane parametry ukladu krzepniecia u chorych z hiperlipidemia typu II.

The studies results of statin influence on hemostasis are various. The aim of our study was to evaluate the effects of simvastatin and atorvastatin on hemostatic parameters, such as activity of factor X, antithrombin III, fibrinogen concentration and Lp(a). 72 patients (pts) aged 40-65 were involved in the study; 49 of them suffered from hyperlipidemia II (hlp II) with the initial concentration of total cholesterol (TC) >200 mg/dL, cholesterol LDL (LDL-C) >145 mg/dL, triglycerides (TG) <400 mg/dL. The control group consisted of 20 healthy persons. The pts with hlp II who underwent 4 weeks long lipid lowering diet were randomized into two groups: I--27 pts treated with simvastatin (20 mg/d), II--22 pts treated with atorvastatin (10 mg/d). The active statin therapy lasted 8 weeks. The activity of factor X and antithrombin III (AT III) was estimated by amidolytic methods, fibrinogen concentration (Fb) by the Clauss method, Lp(a)-immunoenzymatic method. The mean values of factor X activity and Fb serum concentration were higher in pts with hip II than in the control group, the AT III activity was lower. The Lp(a) concentration didn't differ between groups. Statin treatment was associated with significant reduction of factor X activity. Both simvastatin and atorvastatin markedly increased AT III (87%, 98%) in comparison to the initial values. No changes of Lp(a) concentration were observed during statin therapy. Atorvastatin therapy significantly increased the Fb concentration (12.3%). Simvastatin treatment didn't influence Fb concentration.

[Evaluation of thrombin generation and clot bound thrombin in plasma of hyperlipidemic patients treated with statins]. / Ocena stopnia generacji trombiny i wiazania trombiny ze skrzepem w osoczu ludzi z hiperlipidemia typu II leczonych statynami.

Thrombin is a crucial enzyme in blood coagulation cascade having both pro- and antithrombotic properties. Disorders of hemostatic balance increase possibility of clot formation and play significant role in the development of atherosclerosis. Statins applied in prevention of cardiovascular diseases, have not only hypolipemic activity but also many pleiotropic effects. The aim of this study was to evaluate the level of thrombin generation and clot bound thrombin in patients with hyperlipidemia type II (hlpII) before and after statins treatment. 81 patients were involved in this study: 59 patients with hlp II and 22 healthy. Patients with hlp II were treated with pravastatin (20 mg/day; n=10), simvastatin (20 mg/day; n=22), atorvastatin (10 mg/day; n=27). The treatment in each of groups lasted 8 weeks. Thrombin generation and clot bound thrombin level were estimated before and after therapy by means of spectrophotometric method with usage of chromogenic substrate S-2238. Our results demonstrate that therapy with atorva- simva- and pravastatin improves lipid levels in plasma and investigated hemostasis parameters. All three statins statistically significantly decrease total generated thrombin. Atorva- and simvastatin also significantly decrease activity both free generated thrombin and clot bound thrombin. No correlation between lipidogram and hemostatic parameters after treatment with statins suggests that observed changes are pleiotropic effect of statins treatment.

Pseudomonas Exotoxin A: optimized by evolution for effective killing.

Pseudomonas Exotoxin A (PE) is the most toxic virulence factor of the pathogenic bacterium Pseudomonas aeruginosa. This review describes current knowledge about the intoxication pathways of PE. Moreover, PE represents a remarkable example for pathoadaptive evolution, how bacterial molecules have been structurally and functionally optimized under evolutionary pressure to effectively impair and kill their host cells.

Isolation and characterization of collagen from the skin of Brama australis.

Collagen was extracted from the skin of Brama australis, the fish from warm-water sea. The yield of collagen from skin of B. australis was about 1.5% on a wet weight basis of raw material. The isolated protein was confirmed as collagen by different physico-chemical techniques such as: FTIR, SDS-PAGE, and amino acid analysis. The denaturation temperature (T(d)) of obtained collagen was found to be 24°C, what is promising as an advantage for cosmetic application. According to the electrophoretic pattern, collagen consisted of two different α-chains (α1 and α2) and was classified as type I collagen. Although T(d) of obtained collagen is higher than 20 °C it is still far from T(d) of mammalian collagen.