Publications by authors named "Tomasz Przygodzki"

26 Publications

  • Page 1 of 1

Diketopiperazine-Based, Flexible Tadalafil Analogues: Synthesis, Crystal Structures and Biological Activity Profile.

Molecules 2021 Feb 3;26(4). Epub 2021 Feb 3.

Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, 02-089 Warsaw, Poland.

Phosphodiesterase 5 (PDE5) is one of the most extensively studied phosphodiesterases that is highly specific for cyclic-GMP hydrolysis. PDE5 became a target for drug development based on its efficacy for treatment of erectile dysfunction. In the present study, we synthesized four novel analogues of the phosphodiesterase type 5 (PDE5) inhibitor-tadalafil, which differs in (i) ligand flexibility (rigid structure of tadalafil vs. conformational flexibility of newly synthesized compounds), (ii) stereochemistry associated with applied amino acid building blocks, and (iii) substitution with bromine atom in the piperonyl moiety. For both the intermediate and final compounds as well as for the parent molecule, we have established the crystal structures and performed a detailed analysis of their structural features. The initial screening of the cytotoxic effect on 16 different human cancer and non-cancer derived cell lines revealed that in most cases, the parent compound exhibited a stronger cytotoxic effect than new derivatives, except for two cell lines: HEK 293T (derived from a normal embryonic kidney, that expresses a mutant version of SV40 large T antigen) and MCF7 (breast adenocarcinoma). Two independent studies on the inhibition of PDE5 activity, based on both pure enzyme assay and modulation of the release of nitric oxide from platelets under the influence of tadalafil and its analogues revealed that, unlike a reference compound that showed strong PDE5 inhibitory activity, the newly obtained compounds did not have a noticeable effect on PDE5 activity in the range of concentrations tested. Finally, we performed an investigation of the toxicological effect of synthesized compounds on in the highest applied concentration of and (160 μM) and did not find any effect that would suggest disturbance to the life cycle of . The lack of toxicity observed in and enhanced, strengthened selectivity and activity toward the MCF7 cell line made good leading structures for further structure activity optimization and makes a reasonable starting point for the search of new, selective cytotoxic agents.
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http://dx.doi.org/10.3390/molecules26040794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913621PMC
February 2021

Intravital Assessment of Blood Platelet Function. A Review of the Methodological Approaches with Examples of Studies of Selected Aspects of Blood Platelet Function.

Int J Mol Sci 2020 Nov 6;21(21). Epub 2020 Nov 6.

Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, 92-215 Lodz, Poland.

Platelet biology owes to intravital studies not only a better understanding of platelets' role in primary hemostasis but also findings that platelets are important factors in inflammation and atherosclerosis. Researchers who enter the field of intravital platelet studies may be confused by the heterogeneity of experimental protocols utilized. On the one hand, there are a variety of stimuli used to activate platelet response, and on the other hand there are several approaches to measure the outcome of the activation. A number of possible combinations of activation factors with measurement approaches result in the aforementioned heterogeneity. The aim of this review is to present the most often used protocols in a systematic way depending on the stimulus used to activate platelets. By providing examples of studies performed with each of the protocols, we attempt to explain why a particular combination of stimuli and measurement method was applied to study a given aspect of platelet biology.
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http://dx.doi.org/10.3390/ijms21218334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664321PMC
November 2020

Fibrinogen Glycation and Presence of Glucose Impair Fibrin Polymerization-An In Vitro Study of Isolated Fibrinogen and Plasma from Patients with Diabetes Mellitus.

Biomolecules 2020 06 7;10(6). Epub 2020 Jun 7.

Department of Hemostasis and Hemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, Mazowiecka 6/8, 92-216 Lodz, Poland.

Background: Fibrin formation and structure may be affected by a plethora of factors, including both genetic and posttranslational modifications, such as glycation, nitration or acetylation.

Methods: The present study examines the effect of fibrinogen glycation on fibrin polymerization, measured in fibrinogen concentration-standardized plasma of subjects with type 2 diabetes mellitus (T2DM) and in a solution of human fibrinogen exposed to 30 mM glucose for four days.

Results: The fibrin polymerization velocity (V) observed in the T2DM plasma (median 0.0056; IQR 0.0049‒0.0061 AU/s) was significantly lower than in non-diabetic plasma (median 0.0063; IQR 0.0058‒0.0071 AU/s) ( < 0.05). Furthermore, significantly lower V was observed for glucose-treated fibrinogen (V 0.046; IQR 0.022‒0.085 AU/s) compared to control protein incubated with a pure vehicle (V 0.053; IQR 0.034‒0.097 AU/s) ( < 0.05). The same tendency was observed in the fibrinogen samples supplemented with 6 mM glucose just before measurements. It is assumed that glucose may affect the ability of fibrinogen to form a stable clot in T2DM subjects, and that this impairment is likely to influences the outcomes of some diagnostic assays. As the example, the impaired clotting ability of glycated fibrinogen may considerably influence the results of the standard Clauss method, routinely used to determine fibrinogen concentration in plasma. The stoichiometric analysis demonstrated that spontaneous glycation at both the sites with high and low glycation potential clearly dominated in T2DM individuals in all fibrinogen chains.
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http://dx.doi.org/10.3390/biom10060877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356284PMC
June 2020

data: treatment with the F11R/JAM-A peptide 4D decreases mortality and reduces the generation of atherosclerotic plaques in ApoE-deficient mice.

Data Brief 2020 Jun 23;30:105516. Epub 2020 Apr 23.

Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, New York 11203, USA.

The data in this article focus on the F11 Receptor (F11R/JAM-A; Junctional Adhesion Molecule-A; JAM-A, F11R), a cell adhesion protein constitutively expressed on the membrane surface of circulating platelets and localized within the tight junctions of healthy endothelial cells (ECs). Previous reports have shown that F11R/JAM-A plays a critical role in the adhesion of platelets to an inflamed endothelium due to its' pathological expression on the luminal surface of the cytokine-inflamed endothelium. Since platelet adhesion to an inflamed endothelium is an early step in the development of atherosclerotic plaque formation, and with time, resulting in heart attacks and stroke, we conducted a long-term, study utilizing the atherosclerosis-prone ApoE mice to attempt a blockade of the formation of atherosclerotic plaques by preventing the adhesion of platelets to the inflamed vasculature . Utilizing a nonhydrolyzable peptide derived from an amino acid sequence of F11R/JAM-A, peptide 4D, we have shown that the adhesion of platelets to the inflamed endothelial cells could be blocked by peptide 4D. The present data demonstrate the positive health benefits of chronic peptide 4D administration to the atherosclerosis-prone ApoE mice, and provides new information for potential use of this F11R derived peptide in the prevention of atherosclerosis. The data presented in this article provide further experimental support for the study presented in Babinska et al., Atherosclerosis 284 (2019) 92-101.
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http://dx.doi.org/10.1016/j.dib.2020.105516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206208PMC
June 2020

Diabetes and Hyperglycemia Affect Platelet GPIIIa Expression. Effects on Adhesion Potential of Blood Platelets from Diabetic Patients under In Vitro Flow Conditions.

Int J Mol Sci 2020 May 2;21(9). Epub 2020 May 2.

Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland.

Blood platelets play a crucial role in the early stages of atherosclerosis development. The process is believed to require firm adhesion of platelets to atherosclerosis-prone sites of the artery. However, little evidence exists regarding whether the blood platelets of individuals with pathological conditions associated with atherosclerosis have higher potential for adhesion. This process is to a large extent dependent on receptors present on the platelet membrane. Therefore, the aim of the presented study was to determine whether blood platelets from diabetic patients have higher capacity of adhesion under flow conditions and how diabetes affects one of the crucial platelet receptors involved in the process of adhesion-GPIIIa. The study compares the ability of platelets from non-diabetic and diabetic humans to interact with fibrinogen and von Willebrand factor, two proteins found in abundance on an inflamed endothelium, under flow conditions. The activation and reactivity of the blood platelets were also characterized by flow cytometry. Platelets from diabetic patients did not demonstrate enhanced adhesion to either studied protein, although they presented increased basal activation and responsiveness towards low concentrations of agonists. Platelets from diabetic patients were characterized by lower expression of GPIIIa, most likely due to an enhanced formation of platelet-derived microparticles PMPs, as supported by the observation of elevated concentration of this integrin and of GPIIIa-positive PMPs in plasma. We conclude that altered functionality of blood platelets in diabetes does not increase their adhesive potential. Increased glycation and decrease in the amount of GPIIIa on platelets may be partially responsible for this effect. Therefore, higher frequency of interactions of platelets with the endothelium, which is observed in animal models of diabetes, is caused by other factors. A primary cause may be a dysfunctional vascular wall.
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http://dx.doi.org/10.3390/ijms21093222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247361PMC
May 2020

Adenosine Receptor Agonists Exhibit Anti-Platelet Effects and the Potential to Overcome Resistance to P2Y Receptor Antagonists.

Molecules 2019 Dec 28;25(1). Epub 2019 Dec 28.

Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Faculty of Health Sciences, Medical University of Lodz, Mazowiecka 6/8, 92-235 Lodz, Poland.

Large inter-individual variation in platelet response to endogenous agonists and pharmacological agents, including resistance to antiplatelet therapy, prompts a search for novel platelet inhibitors and development new antithrombotic strategies. The present in vitro study evaluates the beneficial effects of three adenosine receptor (AR) agonists (regadenoson, LUF 5835 and NECA), different in terms of their selectivity for platelet adenosine receptors, when used alone and in combination with P2Y inhibitors, such as cangrelor or prasugrel metabolite. The anti-platelet effects of AR agonists were evaluated in healthy subjects (in the whole group and after stratification of individuals into high- and low-responders to P2Y inhibitors), using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y antagonists, AR agonists were much less or not effective under static conditions, but demonstrated similar antiplatelet activity in flow. In most cases, AR agonists significantly enhanced the anti-platelet effect of P2Y antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y antagonists were similar in high- and low-responders to P2Y inhibitors. In conclusion, a combination of anti-platelet agents acting via the P1 and P2 purinergic receptors represents a promising alternative to existing antithrombotic therapy.
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http://dx.doi.org/10.3390/molecules25010130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982709PMC
December 2019

A peptide antagonist of F11R/JAM-A reduces plaque formation and prolongs survival in an animal model of atherosclerosis.

Atherosclerosis 2019 05 22;284:92-101. Epub 2019 Feb 22.

Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, NY, 11203, USA.

Background And Aims: The F11 Receptor (F11R), AKA Junctional Adhesion Molecule-A (JAM-A) (F11R/JAM-A), is an adhesion protein constitutively expressed on the membrane surface of circulating platelets and the luminal surface of inflamed endothelial cells (EC). Platelet adhesion to an inflamed endothelium is one of the early steps of atherosclerotic plaque formation. Our previous studies, conducted with cultured EC in vitro, have demonstrated the expression of F11R/JAM-A on the luminal surface of inflamed EC, platelet adhesion to inflamed EC through F11R/JAM-A interactions, and inhibition of this interaction by the presence of F11R/JAM-A antagonistic peptide (F11Rpeptide 4D). In the present study, we examined in vivo the overall health-benefits and cardiovascular effects of long-term treatment of animals prone to atherosclerosis, ApoE mice, with F11R-peptide 4D.

Methods: Twenty ApoE mice were assigned to daily treatment with peptide 4D and compared to their counterparts control untreated mice. Mice were observed for wellness and survival. Plaque size in the aorta and heart was measured using histological analysis. Effects of peptide 4D (or scramble control) on platelet adhesion to inflamed endothelium were measured using intravital microscopy.

Results: Significant reductions in atherosclerotic plaques number and size, an overall robust health with longer survival were found in the peptide 4D treated group of ApoE mice. Intravital microscopic studies conducted in exposed vessels of ApoE mice demonstrated significant inhibition by peptide 4D of platelet adhesion to the cytokine-inflamed endothelium.

Conclusions: Our results demonstrate that peptide 4D significantly reduces atherosclerotic plaque formation in ApoE mice and inhibits platelet adhesion to the inflamed arterial endothelium.
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http://dx.doi.org/10.1016/j.atherosclerosis.2019.02.014DOI Listing
May 2019

Effects of three-month streptozotocin-induced diabetes in mice on blood platelet reactivity, COX-1 expression and adhesion potential.

Int J Exp Pathol 2019 02 27;100(1):41-48. Epub 2019 Feb 27.

Department of Haemostatic Disorders, Chair of Biomedical Sciences, Faculty of Health Sciences, Medical University of Lodz, Lodz, Poland.

Diabetes is associated with an increased risk of cardiovascular disease. This is partially attributed to an altered activation status of blood platelets in this disease. Previously, alterations have been shown in COX-1 and protease activated receptor (PAR)-3 receptor expression in platelets in two animal models of diabetes, there have not been studies which address expression of these proteins in mice with long-term streptozotocin (STZ)-induced diabetes. We have also addressed the effect of diabetes on platelet adhesion under flow conditions. With the use of flow cytometry, we have shown that certain markers of platelet basal activation, such as active form of α β and of CD40L were increased in STZ-induced diabetic mice. Platelets from STZ-induced diabetic mice were also more reactive when stimulated with PAR-4 activating peptide as revealed by higher expression of active form of α β , membrane-bound on vWillebrand Factor and binding of exogenous fluorescein isothyanate-labelled fibrinogen. Expression of COX-1 and production of thromboxane A in platelets of STZ-induced diabetic mice were higher than in control animals. We observed no effect of diabetes on ability of platelets to form stable adhesions with fibrinogen in flow conditions. We conclude that although certain similarities exist between patterns of activation of platelets in animal models of diabetes, the differences should also be taken into account.
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http://dx.doi.org/10.1111/iep.12298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463392PMC
February 2019

Comparison of different microscopy approaches to quantification of inhibitory effect on thrombus formation under flow conditions by the example of adenosine receptor agonist HE-NECA.

J Pharmacol Toxicol Methods 2018 Nov - Dec;94(Pt 1):94-104. Epub 2018 Jul 19.

Department of Haemostatic Disorders, Chair of Biomedical Sciences, Faculty of Health Sciences, Medical University of Lodz, 6/8 Mazowiecka Street, 92-235 Lodz, Poland.

Introduction: Thrombus formation in vitro in flow conditions and its visualization and quantification with the use of microscopy are widely utilized to evaluate activity of compounds with a potential antithrombotic activity. Visualization and quantification of thrombi can be performed with the use of wide-field or confocal microscopy. Acquiring reliable numerical data from wide-field microscopy images of objects which have a complex three-dimensional structure is strongly influenced by the methods used for image analysis. This can be a possible source of inaccuracy in assessment of antithrombotic activity of a tested substance. We aimed to verify how different approaches to the quantification of wide-field images can affect the evaluation of an antiplatelet effect of a tested substance.

Methods: We compared three algorithms of image analysis to evaluate an effect of 2-hexynyl-5'-ethylcarboxamidoadenosine (HE-NECA), a compound of a moderate antiplatelet activity on thrombus formation, and of abciximab - a potent antiplatelet compound. Also, we studied how the results obtained in a wide-field imaging correspond to those obtained by means of confocal imaging.

Results: Three algorithms for analysis of wide-field images showed antiplatelet effect of HE-NECA or abciximab. Absolute values of thrombus area and outcomes of the evaluation of inhibition efficacy of HE-NECA were significantly different between the algorithms. Analysis of volumes and heights of thrombi obtained by confocal imaging confirmed inhibitory effect of HE-NECA, but the evaluated levels of inhibition were significantly different from that obtained by wide-field imaging.

Discussion: We conclude that wide-field imaging provides reliable qualitative data on an inhibitory effect on thrombus formation, despite differences which can emerge from various approaches to image analysis. However, quantitative evaluation and comparison of the efficacy of inhibitors on the basis of total area occupied by thrombi obtained by wide-field microscopy should be made with caution. To obtain a reliable quantitative assessment of the effect of a tested compound on thrombus structure the use of confocal microscopy is inevitable.
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http://dx.doi.org/10.1016/j.vascn.2018.07.003DOI Listing
December 2018

Enhanced adhesion of blood platelets to intact endothelium of mesenteric vascular bed in mice with streptozotocin-induced diabetes is mediated by an up-regulated endothelial surface deposition of VWF - In vivo study.

Platelets 2018 Jul 26;29(5):476-485. Epub 2017 Jul 26.

a Department of Haemostasis and Haemostatic Disorders , Medical University of Lodz , Lodz , Poland.

Numerous in vitro experiments have confirmed that a dysfunctional endothelium is characterized by, inter alia, a higher affinity for binding of platelets and leukocytes. However, there is still no direct evidence for greater interaction between platelets and intact endothelium in in vivo animal models of diabetes. Therefore, the present study examines the pro-adhesive properties of endothelium change in vivo as an effect of streptozotocin (STZ)-induced diabetes and the role of two key platelet receptors: GPIb-IX-V and GPIIb/IIIa. Mice of C57BL strain with streptozotocin-induced diabetes were used in the study. Flow cytometry was used to assess basal activation and reactivity of platelets. Adhesion of platelets to the vascular wall was visualized with the use of intravital microscopy in mesentery. The contribution of GPIIb/IIIa and GPIb-IX-V was evaluated by the injection of Fab fragments of respective antibodies. The integrity of the endothelium and vWf expression were evaluated histochemically. Basal activation and reactivity of platelets in streptozotocin-diabetic mice were elevated. Blood platelets adhered more often to the vascular wall of diabetic mice than nondiabetic animals: 11.9 (6.4; 32.8) plt/min/mm (median [IQR]) vs 2.7 (1.3; 6.4) plt/min/mm. The injection of anti-GPIbα antibodies decreased the number of adhering platelets from 89.5 (34.0; 113.1) plt/min/mm (median [IQR]) in mice treated with isotype antibodies to 3.1 (1.7; 5.6) plt/min/mm in mice treated with blocking antibodies. The effect of GPIIb/IIIa blockage was not significant. Immunohistochemistry revealed a higher expression of vWF in the endothelium of STZ mice, but no substantial changes in endothelial morphology were detected. To conclude, the study shows that the platelets interact more frequently with the mesenteric vascular bed in mice with 1-month STZ-induced diabetes than in healthy mice. These interactions are mediated via platelet GPIb-IX-V and are driven by increased expression of vWF in endothelial cells.
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http://dx.doi.org/10.1080/09537104.2017.1332365DOI Listing
July 2018

Flow cytometry analysis reveals different activation profiles of thrombin- or TRAP-stimulated platelets in db/db mice. The regulatory role of PAR-3.

Blood Cells Mol Dis 2017 06 22;65:16-22. Epub 2017 Mar 22.

Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, 6/8 Mazowiecka str., 92-215 Lodz, Poland.

Introduction: Recent studies have shown that it may be the concentration of thrombin, which is discriminative in determining of the mechanism of platelet activation via protease activated receptors (PARs). Whether the observed phenomenon of differentiated responses of mouse platelets to various thrombin concentrations in non-diabetic db/+ and diabetic db/db mice depends upon the concerted action of various PARs, remains to be established.

Results: We found elevated reactivity of platelets, as well as the enhanced PAR-3 expression in response to both the used concentrations of AYPGKF in db/db mice, as compared to db/+ heterozygotes. At low concentration of thrombin platelets from diabetic mice demonstrated hyperreactivity, reflected by higher expression of PAR-3. For higher thrombin concentration, blood platelets from db/db mice appeared hyporeactive, compared to db/+ animals, while no significant differences in PAR-3 expression were observed between diabetic and non-diabetic mice.

Conclusions: The novel and previously unreported finding resulting from our study is that the increased expression of PAR-3 in response to either TRAP for PAR-4 or low thrombin (when PAR-4 is not the efficient thrombin receptor) may be one of the key events contributing to higher reactivity of platelets in db/db mice.
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http://dx.doi.org/10.1016/j.bcmd.2017.03.011DOI Listing
June 2017

Quantification of the Blood Platelet Reactivity in the ADP-Induced Model of Non-Lethal Pulmonary Thromboembolism in Mice with the Use of Laser Doppler Flowmetry.

PLoS One 2016 11;11(1):e0146346. Epub 2016 Jan 11.

Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, Lodz, Poland.

Introduction: The paper describes an alternative method for quantification of in vivo ADP-induced thromboembolism. The aim of the studies was to develop a method of quantification which would not require either extravasation or labelling of platelets. Our proposed approach is based on the monitoring of changes of blood flow with the use of laser Doppler flowmetry.

Materials And Methods: Mice of C57Bl strain were used in the study. ADP was injected to the vena cava and blood flow was monitored with the use of a laser Doppler flowmeter in the mesentery. Measurements in platelet-depleted mice, mice pretreated with cangrelor, an ADP receptor antagonist, and eptifibatide, a blocker of fibrinogen binding to GPIIbIIIa, were conducted as the proof-of-concept in the performed experiments. Intravital microscopy and ex vivo imaging of organs was performed to identify the sites of aggregate formation resulting from ADP injection.

Results: The injection of ADP resulted in a dose-dependent reduction of the blood flow in the mesentery. These responses were fully attributable to blood platelet aggregation, as shown by the lack of the effect in platelet-depleted mice, and significantly reduced responses in mice pretreated with cangrelor and eptifibatide. No platelet aggregate formation in mesenteric vessels was revealed by intravital microscopy, while ex vivo imaging showed accumulation of fluorescent labelled platelets in the lung.

Conclusions: Injection of ADP to the venous system results in the formation of platelet aggregates predominantly in the lung. This results in reversible blood flow cessation in peripheral blood vessels. The measurement of this blood flow cessation in the mesentery allows indirect measurement of ADP-induced pulmonary thromboembolism. We suggest that this approach can be useful for in vivo screening for antiplatelet drug candidates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146346PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713441PMC
July 2016

How do the full-generation poly(amido)amine (PAMAM) dendrimers activate blood platelets? Activation of circulating platelets and formation of "fibrinogen aggregates" in the presence of polycations.

Int J Pharm 2016 Apr 28;503(1-2):247-61. Epub 2015 Aug 28.

University of Lodz, Faculty of Biology and Environmental Protection, Department of Thermobiology, Pomorska 141/143, 90-236 Lodz, Poland.

Direct use of poly(amido)amine (PAMAM) dendrimers as drugs may be limited, due to uncertain (cyto)toxicity. Peripheral blood components, which constitute the first line of a contact with administered pharmaceuticals, may become vastly affected by PAMAM dendrimers. The aim of this study was to explore how PAMAMs' polycationicity might affect blood platelet activation and reactivity, and thus trigger various haemostatic events. We monitored blood platelet reactivity in rats with experimental diabetes upon a long-term administration of the unmodified PAMAM dendrimers. In parallel, the effects on blood flow in a systemic circulation was recorded intravitally in mice administered with PAMAM G2, G3 or G4. Compounding was the in vitro approach to monitor the impact of PAMAM dendrimers on blood platelet activation and reactivity and on selected haemostatic and protein conformation parameters. We demonstrated the activating effects of polycations on blood platelets. Some diversity of the revealed outcomes considerably depended on the used approach and the particular technique employed to monitor blood platelet function. We discovered undesirable impact of plain PAMAM dendrimers on primary haemostasis and their prothrombotic influence. We emphasize the need of a more profound verifying of all the promising findings collected for PAMAMs with the use of well-designed in vivo preclinical studies.
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http://dx.doi.org/10.1016/j.ijpharm.2015.08.073DOI Listing
April 2016

Inhibition of cyclooxygenase-2 causes a decrease in coronary flow in diabetic mice. The possible role of PGE2 and dysfunctional vasodilation mediated by prostacyclin receptor.

J Physiol Biochem 2015 Sep 5;71(3):351-8. Epub 2015 May 5.

Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, Lodz, 92-215, Poland,

Several lines of evidence suggest that cyclooxygenase-2 (COX-2) activity can have a beneficial role in the maintenance of vascular tone of the blood vessels in diabetes. Specifically, the increased production of prostacyclin (PGI2) and prostaglandin E2 (PGE2), mediated by COX-2, has been suggested to compensate for decreased synthesis of nitric oxide (NO). The study investigates whether inhibition of COX-2 may reduce the coronary flow in diabetic animals and may also lead to decreased synthesis of prostaglandins. Mice aged 18-20 weeks were used for the study: those with leptin receptor deficiency (db/db) served as a model of diabetes while heterozygous (db/+) mice served as controls. Coronary flow was measured by the Langendorff method, and prostaglandin synthesis by myocardia was assayed in heart perfusates. COX-2 inhibition was found to reduce basal coronary flow in db/db mice but had no effect in db/+ mice. Secretion of PGE2 was found to be higher in db/db mice, while prostacyclin synthesis did not differ. COX-2 inhibition decreased production of both prostaglandins to similar levels in both groups. The use of ONO-1301, a specific agonist for the prostacyclin receptor revealed that vasodilating responses mediated by the receptor were impaired in db/db mice. The expression levels of the receptor in cardiac tissue did not differ between the groups. It is concluded that the increased COX-2 contribution to vasodilation in diabetic animals appears to be partially a result of increased COX-2-dependent synthesis of PGE2 and also may be caused by impaired vasodilation mediated by the prostacyclin receptor.
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http://dx.doi.org/10.1007/s13105-015-0415-yDOI Listing
September 2015

β-Resorcylidene aminoguanidine (RAG) dilates coronary arteries in an endothelium-independent manner.

Pharmacol Rep 2015 Jun 20;67(3):631-5. Epub 2015 Jan 20.

Department of Haemostatic Disorders, Medical University of Lodz, Łódź, Poland.

Background: β-Resorcylidene aminoguanidine (RAG), a highly reactive derivative of aminoguanidine, possesses antithrombotic activity which involves the activation of the vascular COX-2/PGI2 pathway. This endothelium-dependent effect suggests that RAG may demonstrate vasomotor activity in arterial vessels. The aim of the present study was to investigate a possible vasoactive action of RAG in coronary arteries of rat heart.

Methods: Isolated rat hearts were perfused in the Langendorff model. To investigate the dose dependency of the effect of RAG on coronary flow, the hearts were perfused with RAG at increasing concentrations. Mechanisms of RAG-mediated vasodilation were subsequently tested using selective inhibitors of the endothelium-dependent and endothelium-independent mechanisms responsible for regulation of vascular tone.

Results: RAG dilated coronary arteries at concentrations above 10(-5)mol/l. Inhibition of the endothelium-dependent mechanism of vasodilation by NG-nitro-L-arginine methyl ester, indomethacin and aminobenzotriazole did not affect RAG-mediated vasodilation. Other compounds also had no impact on the vasodilating effect of RAG: the NO-dependent guanylate cyclase inhibitor - 1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one, the cAMP-dependent protein kinase inhibitor - PKAi, and the K(+) channel blockers - glibenclamide, tetraethylammonium, charybdotoxin, and apamin.

Conclusions: RAG is a strong vasodilator that exerts its effect via endothelium-independent mechanisms.
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http://dx.doi.org/10.1016/j.pharep.2015.01.003DOI Listing
June 2015

Can metabolic impairments in experimental diabetes be cured with poly(amido)amine (PAMAM) G4 dendrimers? In the search for minimizing of the adverse effects of PAMAM administration.

Int J Pharm 2014 Apr 21;464(1-2):152-67. Epub 2014 Jan 21.

Department of Haemostasis and Haemostatic Disorders, Medical University of Lodz, University Clinical Hospital No. 2, Zeromskiego 113, 90-549 Lodz, Poland. Electronic address:

Poly(amido)amine (PAMAM) G4 dendrimers, given intraperitoneally to diabetic rats, have been reported to scavenge excessive blood glucose and minimize the effects of hyperglycaemia, however, at the cost of reduced survival. This paper is the first to compare the effectiveness of three different routes of PAMAM G4 administration with regard to minimizing the adverse effects of hyperglycaemia in rats. Hence, the aim of the study is to identify the most effective and the least harmful method of dendrimer administration. Control and streptozotocin-diabetic Sprague-Dawley rats were exposed to PAMAM G4 (0.5 μmol/kg b.w.) for 60 days, administered intraperitoneally, intragastrically or subcutaneously. Intraperitoneal and subcutaneous administration of PAMAM G4 was found to be most effective in suppressing the long-term markers of hyperglycaemia, while the intragastric route appeared the least effective. Otherwise, the greatest incidence of adverse effects was associated with intraperitoneal and the lowest with subcutaneous delivery. Harmful effects of intragastrical administration were much lower compared to intraperitoneal route, but at the cost of reduced hypoglycaemizing potential. Otherwise, subcutaneous injection represents the best compromise of moderate PAMAM dendrimer toxicity and effective reduction in the markers of long-term severe hyperglycaemia in chronic experimental diabetes.
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http://dx.doi.org/10.1016/j.ijpharm.2014.01.011DOI Listing
April 2014

COX-2-derived prostaglandins do not contribute to coronary flow regulation in diabetic rats: distinct secretion patterns of PGI2 and PGE2.

Eur J Pharmacol 2013 Jan 28;700(1-3):86-92. Epub 2012 Dec 28.

Department of Haemostasis and Haemostatic Disorders, Chair of Laboratory Diagnostics, Medical University of Lodz, Central Veterans' Hospital, Lodz, Poland.

The role of cyclooxygenase-2 (COX-2) in restoring the functions of impaired endothelium is attracting considerable attention, notably the function of COX-2-derived vasodilatory prostaglandins is disputed in the context of the regulation function in the impaired vascular beds. We have examined the hypothesis that COX-2 activity contributes more to vasodilation in hyperglycemic animals than in healthy counterparts, and that COX-2 derived vasodilatory prostaglandins (PGI(2) and PGE(2)) are responsible for this effect. Using the Langendorff heart perfusion system, the effects of COX-2 inhibition were monitored on both basal and bradykinin-induced coronary flows in Sprague-Dawley rats given 8-week streptozotocin-induced diabetes and in age-matched controls (n=15). Secretions of PGI(2) and PGE(2), both total and the COX-2 dependent pools, have also been compared. The selective COX-2 inhibitor, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398), had no effect on coronary flow in the diabetic group of animals. Thus, the compensatory role of COX-2 in regulation of vascular tone in experimental diabetes found in other experimental models was not confirmed. However, COX-2 activity significantly contributed to PGI(2) synthesis in healthy rats, with prostacyclin secretion being two-fold decreased by NS-398. Contrary to our hypothesis, neither prostacyclin nor PGE(2) production differed between the experimental groups under the basal conditions. Bradykinin had no effect on the secretion of PGI(2) in either group, but increased PGE(2) synthesis in healthy animals, although not in the streptozotocin group. PGE(2) production in response to bradykinin was COX-2-dependent in control animals. We conclude that, in rats with 8-week streptozotocin-induced diabetes, the activity of COX-2 in coronary vasculature is not significantly enhanced.
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http://dx.doi.org/10.1016/j.ejphar.2012.12.026DOI Listing
January 2013

N-Methyl-2-pyridone-5-carboxamide is 1-methylnicotinamide metabolite of low cyclooxygenase-dependent vasodilating activity.

J Physiol Biochem 2012 Sep 13;68(3):329-34. Epub 2012 Jan 13.

Chair of Laboratory Diagnostics, Department of Haemostasis and Haemostatic Disorders, University Clinical Hospital No 2, Medical University of Lodz, Lodz, Poland.

1-Methylnicotinamide (MNA) is a primary metabolite of nicotinamide recently proven to cause systemic increase in PGI(2) plasma levels in an unknown mechanism. Our present study was aimed at verifying whether the increased production of PGI(2), a vasodilating prostanoid, in response to MNA, its metabolite N-methyl-2-pyridone-5-carboxamide (Met2PY), and nicotinamide may be reproduced under in vitro conditions. Since prostacyclin is a vasodilating prostanoid, we also performed the functional tests in the ex vivo model of coronary vascular bed perfusion to evaluate the vasoactive properties of those compounds. We did not observe any significant effect of the tested drugs on either PGI(2) or PGE(2) secretion in our in vitro model. Nicotinamide at the concentrations of 10 and 100 μmol/l and 100 μmol/l Met2PY slightly but significantly increased coronary flow in rat heart. These increases, however, remained very low when compared to that induced by the reference compound, bradykinin (100 nmol/l). Perfusion of rat hearts with Met2PY in the presence of 50 μmol/l indomethacin resulted in decreased coronary flow, which proves that the effect is cyclooxygenase dependent. We conclude that MNA metabolites should be more carefully addressed in reference to pro-prostacyclin activity and that systemic mechanism of MNA-induced PGI(2) production needs further clarification.
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http://dx.doi.org/10.1007/s13105-012-0144-4DOI Listing
September 2012

Effects of 1-methylnicotinamide and its metabolite N-methyl-2-pyridone-5-carboxamide on streptozotocin-induced toxicity in murine insulinoma MIN6 cell line.

Acta Biochim Pol 2011 14;58(1):75-7. Epub 2011 Mar 14.

Department of Haemostasis and Haemostatic Disorders, Chair of Laboratory Diagnostics, Medical University of Lodz, University Clinical Hospital No. 2, Łódź, Poland.

1-methylnicotinamide (MNA) is a primary metabolite of nicotinamide. In recent years several activities of MNA have been described, such as anti-inflammatory activity in skin diseases, induction of prostacyclin synthesis via COX-2, aortal endothelium protection in diabetes and hypertriglyceridaemia and increased survival rate of diabetic rats. 1-methylnicotinamide was also suggested to protect pancreatic cells from streptozotocin in vivo. Streptozotocin toxicity is known to be mediated by poly-ADP-ribose polymerase. Nicotinamide and its derivatives have been shown to ameliorate poly-ADP-ribose polymerase-dependent nucleotide pool reduction. We aimed to verify if 1-methylnicotinamide and its metabolite, N-methyl-2-pyridone-5-carboxamide, can protect insulinoma cells from streptozotocin-induced toxicity. We found that N-methyl-2-pyridone-5-carboxamide, but not 1-methylnicotinamide, restores the pool of ATP and NAD+ in streptozotocin-treated cells, but neither compound improved the cell viability. We conclude that inhibition of poly-ADP-ribose polymerase-dependent nucleotide pool reduction may not be sufficient to protect cells from streptozotocin toxicity.
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July 2011

1-methylnicotinamide effects on the selected markers of endothelial function, inflammation and haemostasis in diabetic rats.

Eur J Pharmacol 2010 Aug 25;640(1-3):157-62. Epub 2010 May 25.

Department of Haemostasis and Haemostatic Disorders, Chair of Laboratory Diagnostics, Medical University of Lodz, University Clinical Hospital No 2, Lodz, Poland.

1-methylnicotinamide (MNA) is a primary metabolite of nicotinamide. In recent years several activities of MNA have been described, such as anti-inflammatory activity in skin diseases, induction of prostacyclin synthesis via COX-2, aortal endothelium protection in diabetes and hypertriglyceridaemia and increasing survival rate of diabetic rats. The aim of the present study was to verify whether the increased survival rate of diabetic animals could be explained by anti-hyperglycaemic activity of MNA and/or by its protective effects on vascular endothelium. We used Sprague-Dawley male rats with an experimental streptozotocin diabetes. The animals received either MNA or pure drinking water. At the particular time intervals groups of rats were sacrificed and the blood was collected. We have shown that MNA increases levels of PGI2 in diabetic rats, but the effect is limited only to the early stage of diabetes. We were unable to prove anti-inflammatory effects of MNA, as it did not affect increased TNF-alpha in diabetic animals. We have confirmed our previous observations that MNA improved survival of diabetic animals, but contrary to our previous study, this effect was not accompanied by improvement in the parameters of long-term glycaemic control. Overall, we conclude that anti-diabetic activity of MNA manifested in the improved lifespan of diabetic animals is rather due to MNA pro-prostacyclin activity, and it may not be substantially related to glycaemic control in diabetes. Still other potential mechanism(s) await further elucidation.
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http://dx.doi.org/10.1016/j.ejphar.2010.05.014DOI Listing
August 2010

Anti-diabetic effects of 1-methylnicotinamide (MNA) in streptozocin-induced diabetes in rats.

Pharmacol Rep 2009 Jan-Feb;61(1):86-98

Department of Hemostasis and Hemostatic Disorders, Chair of Laboratory Diagnostics, Medical University of Łódź, University Clinical Hospital No 2, Poland.

1-Methylnicotinamide (MNA), a major endogenous metabolite of nicotinamide, possesses anti-thrombotic and anti-inflammatory activity, and reverses endothelial dysfunction. In the present work, we investigated whether such a vasoprotective profile of MNA activity affords anti-diabetic action in rats. Diabetes was induced by streptozotocin (STZ) in Sprague-Dawley rats. Eight weeks after STZ injection in untreated or MNA-treated rats (100 mg kg(-1) daily), development of diabetes (plasma concentrations of fasting and non-fasting glucose, HbA(1c), peptide C), development of oxidant stress (lipid peroxidation, carbonylation of plasma proteins), as well as NO-dependent endothelial function in aorta, coronary and mesenteric vessels were analyzed. Finally, the effect of chronic treatment with MNA on long-term survival of diabetic rats was determined. Chronic treatment with MNA profoundly lowered fasting glucose concentrations in plasma, displayed mild effects on plasma HbA(1c) and peptide C concentrations, while having no effects on non-fasting glucose. On the other hand, MNA treatment considerably lowered lipid peroxidation, protein carbonylation, completely prevented impairment of endothelium-dependent vasodilatation in the aorta that was mediated entirely by NO, but failed to affect endothelial function in resistant vessels, which was mediated only partially by NO. Most importantly, chronic treatment with MNA prolonged the long-term survival of diabetic rats. In conclusion, MNA displayed a significant anti-diabetic effect that may be linked to its vasoprotective activity.
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http://dx.doi.org/10.1016/s1734-1140(09)70010-6DOI Listing
June 2009

Effects of resorcylidene aminoguanidine (RAG) on selected parameters of isolated rat liver mitochondria.

Chem Biol Interact 2009 May 18;179(2-3):280-7. Epub 2008 Nov 18.

Department of General Biophysics, University of Lodz, 12/16 Banacha Str., 90-237 Lodz, Poland.

In the present investigation, we attempted to study possible mechanisms of the interactions of resorcylidene aminoguanidine (RAG), the agent with a recognized anti-glycation and antioxidative activity, with rat liver mitochondria. We hypothesized that RAG affects organization of the lipid bilayer in mitochondrial membranes and thus impairs transmembrane Ca(2+) redistribution, transmembrane potential, and respiration capacity. Isolated mitochondria were exposed to RAG (50-200 microM) and several parameters of their function monitored employing spectrofluorimetric, cytometric, and respirometric techniques. Mitochondrial membrane potential and membrane fluidity were tracked using the staining with rhodamine 123 (Rh123) and 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. Mitochondrial respiration and oxidative phosphorylation was monitored with a high-resolution respirometry, and mobilization of Ca(2+) was detected using spectrofluorimetry with Calcium Green 5-N. RAG depolarized and fluidized mitochondrial membrane, as deduced from reduced fluorescence of intramitochondrial Rh123 and decreased DPH fluorescence anisotropy. The slight inhibitory effect of 100-200 microM RAG on mitochondrial respiratory capacity was observed merely when monitored in the presence of ADP. The reduced sensitivity of mitochondria to calcium-induced depolarization was significant only at higher RAG concentrations (100-200 microM). Moreover, RAG induced pronounced conformational changes in two model proteins: bovine serum albumin and cytochrome c. These findings indicate that regardless of its depolarizing and fluidizing properties, RAG does not largely affect the mitochondrial respiration, although it may significantly lower oxidative phosphorylation when used at higher concentrations.
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http://dx.doi.org/10.1016/j.cbi.2008.11.005DOI Listing
May 2009

A pharmacological solution for a conspecific conflict: ROS-mediated territorial aggression in sea anemones.

Toxicon 2008 May 8;51(6):1038-50. Epub 2008 Feb 8.

Department of Biophysics, University of Lodz, Banacha 12/16, 90237 Lodz, Poland.

Venomous organisms are usually resistant to their own venoms, and utilize mechanical behavioral means to resolve intra-specific conflicts, such as those occurring over territory, mates or social status. The present study deals with a venom apparatus, which has been specifically designed for conspecific aggression, by the aid of a unique pharmacology. Actinarian sea anemones such as Actinia equina utilize vesicular organs termed acrorhagi in order to deter conspecific territorial competitors. The territorial aggression was shown to be performed by the aid of acrorhagial cnidocysts, which inflict localized tissue necroses on the body of the approaching-threatening anemone. In view of the fact that sea anemones were shown to resist mechanical injuries and their own cytolytic, necrosis-inducing pore-forming substances-the above acrorhagial injuries are ambiguous. Using an electrical device to collect acrorhagial cnidocyst-derived venom, we have shown that the venom is devoid of paralytic-neurotoxic activity, contains heat denaturable hemolytic polypeptides of a low molecular weight and is capable of inducing intracellular formation of reactive oxygen species (ROS) upon medium application to various cultured cells. The ROS formation phenomenon provides a reasonable pharmacological solution to the, above-mentioned, paradoxical conspecific self-intoxication by triggering a preexisting global endogenous mechanism of oxygen toxicity common to aerobic organisms.
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http://dx.doi.org/10.1016/j.toxicon.2008.01.017DOI Listing
May 2008

Calcium ionophore A23187 action on cardiac myocytes is accompanied by enhanced production of reactive oxygen species.

Biochim Biophys Acta 2005 Jun 9;1740(3):481-8. Epub 2005 Apr 9.

Department of General Biophysics, University of Lodz, Poland.

We show that rat neonatal cardiac myocytes exposed to 1 micromol/l of the calcium ionophore A23187 respond with an enhanced production of reactive oxygen species (ROS). This dose is not cytotoxic to the myocytes. A higher concentration (10 micromol/l) evokes less ROS production and is significantly cytotoxic 24 h after exposure, but not immediately after removal of the A23187, when ROS are measured. Both cell death and the decrease in mitochondrial potential are only partially sensitive to MPT inhibitor cyclosporin A. Experiments performed to elucidate the sources of ROS included use of the nitric oxide synthase (NOS) inhibitor L-NAME; NOS involvement was excluded. Experiments with the oxidative phosphorylation uncoupler CCCP revealed that mitochondria are at least partially responsible for the observed effect. Further studies with cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors (indomethacin and MK886, respectively) showed that these enzymes could also be sources of ROS when the calcium level is elevated. Their effect appeared to be independent of phospholipase A(2) inhibition, suggesting that COX and LOX stimulation is not due to elevated substrate (arachidonic acid) concentration but rather to a direct effect of calcium.
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http://dx.doi.org/10.1016/j.bbadis.2005.03.009DOI Listing
June 2005

Induction of apoptosis and modulation of production of reactive oxygen species in human endothelial cells by diphenyleneiodonium.

Biochem Pharmacol 2005 Apr;69(8):1263-73

Department of Molecular Biophysics, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland.

Diphenyleneiodonium (DPI) inhibits activity of flavoenzymes like NADPH oxidase, the major source of superoxide anion in cardiovascular system, but affects also other oxidoreductases. Contradictory data have been published concerning the effect of diphenyleneiodonium on the production of reactive oxygen species in cells, both inhibitory and stimulatory action of DPI being reported. We have examined the effect of DPI on the cellular production of reactive oxygen and nitrogen species (ROS/RNS) and on the proliferation and apoptosis of human vascular endothelial cells. We found increased oxidation of ROS-sensitive probes (dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate) when DPI (20 microM-100 microM) was present in the treated cells. However, oxidation of the fluorogenic probes was inhibited if DPI (20 microM-100 microM) was removed from the reaction medium after cell preincubation. These results suggest an artifactual oxidation of the fluorogenic probes by DPI or its metabolites. A similar pattern of influence of DPI on the production of NO (measured with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) was observed. Modulation of generation of reactive oxygen and nitrogen species in DPI-treated cells influenced the nitration of tyrosine residues of cellular proteins, estimated by Western blotting. Decreased level of nitration generally paralleled the lowered production of ROS. A decreased 3-(4,5-dimethylthiazolyl)-3-3(4-sulphophenyl) tetrazolium (MTT) reducing activity of cells for was observed immediately after 1h treatment of human endothelial cells with DPI (1 microM-100 microM), in spite of lack of changes in cell viability estimated by other methods. These results point to a next limitation of MTT in estimation of viability of cells treated with oxidoreductase inhibitors. DPI inhibited the proliferation of HUVECs as well as immortalized cell line HUVEC-ST, as assessed by acid phosphatase activity test and measurement of total nucleic acid content. Proapoptotic action of DPI was observed 12 h after incubation with this compound.
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http://dx.doi.org/10.1016/j.bcp.2005.01.010DOI Listing
April 2005

[Therapeutic applications of dendrimers].

Postepy Biochem 2003 ;49(4):290-7

Katedra Biofizyki Ogólnej Uniwersytetu Łódzkiego, ul. S. Banacha 12/16, 90-237 Łódź.

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June 2004