Kornél Miszti-Blasius

The emerging value of P-selectin as a disease marker

Abstract Activated platelets are key components in many arterial disorders. P-selectin is an activation-dependent platelet receptor, which is also identified in endothelial cells. Together with E-and L-selectin it constitutes the selectin family. These transmembrane proteins have continued to attract great interest as they support rapid and reversible cell adhesion in flow systems and thus play an essential role in multicellular interactions during thrombosis and inflammation. Similarly to other lectins, selectins bind to different glycoconjugates with varying affinities. Protein ligands, equipped with the appropriate carbohydrate and sulfate moieties for P-selectin binding, have been identified in normal peripheral blood leukocytes and several non-hematopoietic organs, as well as on cancer cells. For diagnostic purposes, P-selectin can readily be detected on the platelet surface by flow cytometry and by ELISA as a soluble ligand in the plasma. Along with other markers, these data can be used in the assessment of platelet activation status. Such results bear clinical significance since P-selectin has been implicated in the pathogenesis of widespread disorders including coronary artery disease, stroke, diabetes and malignancy.

In vivo application of a small molecular weight antifungal protein of Penicillium chrysogenum (PAF)

The antifungal protein of Penicillium chrysogenum (PAF) inhibits the growth of important pathogenic filamentous fungi, including members of the Aspergillus family and some dermatophytes. Furthermore, PAF was proven to have no toxic effects on mammalian cells in vitro. To prove that PAF could be safely used in therapy, experiments were carried out to investigate its in vivo effects. Adult mice were inoculated with PAF intranasally in different concentrations, up to 2700 μg·kg⁻¹ daily, for 2 weeks. Even at the highest concentration--a concentration highly toxic in vitro for all affected molds used, animals neither died due to the treatment nor were any side effects observed. Histological examinations did not find pathological reactions in the liver, in the kidney, and in the lungs. Mass spectrometry confirmed that a measurable amount of PAF was accumulated in the lungs after the treatment. Lung tissue extracts from PAF treated mice exerted significant antifungal activity. Small-animal positron emission tomography revealed that neither the application of physiological saline nor that of PAF induced any inflammation while the positive control lipopolysaccharide did. The effect of the drug on the skin was examined in an irritative dermatitis model where the change in the thickness of the ears following PAF application was found to be the same as in control and significantly less than when treated with phorbol-12-myristate-13-acetate used as positive control. Since no toxic effects of PAF were found in intranasal application, our result is the first step for introducing PAF as potential antifungal drug in therapy.

Potential Therapeutic Targeting of Platelet-Mediated Cellular Interactions in Atherosclerosis and Inflammation

Cellular interactions among platelets, leukocytes and endothelial cells are considered as a major cause of inflammation and atherosclerosis in many diseases. Via exposed surface receptors and released soluble substances, activated platelets play a crucial role in the initiation of inflammatory processes, resulting in endothelial injury and leading to formation of atherosclerotic plaque with possible thrombotic complications. Classic anti-platelet treatments (e.g. cyclooxygenase inhibitor or ADP-receptor antagonist) have favorable effects in patients with vascular diseases, but they also have several limitations such as increased bleeding risk or non-responsiveness. Thus, the need and opportunities for developing novel therapeutic inhibitors for platelet-mediated events are obvious. Animal and (pre)clinical human studies have suggested that some recently produced specific antagonists of P-selectin from α-granules, as well as its main ligand/receptor P-selectin Glycoprotein Ligand-1, the two major platelet chemokines CXCL4 and CCL5, as well as CD40L, may be considered potential new candidates in the treatment of atherogenesis and inflammation. In this review, we summarize the pathophysiological roles of these effectors in platelet activation and acute or chronic inflammation, and discuss the latest findings on promising antagonistic agents in basic and clinical studies in the prevention of platelet-mediated cellular interactions.

Lack of P-selectin glycoprotein ligand-1 protects mice from thrombosis after collagen/epinephrine challenge.

INTRODUCTION In thrombotic processes, during the association of leukocytes with platelets and endothelial cells, P-selectin glycoprotein ligand-1 (PSGL-1) binds to P-selectin, expressed on activated platelets and endothelial cells. Our aim was to establish the role of PSGL-1 in thrombus formation by evaluating the response to thrombotic stimuli in wild type and PSGL-1 knockout mice. MATERIALS AND METHODS Mice were challenged by tail vein injection of (i) 15 μg collagen plus 3 μg epinephrine (coll/epi) (ii) 7.5 μg collagen plus 1.5 μg epinephrine or (iii) saline. Retro-orbital blood samples were collected in ACD anticoagulaed tubes and platelet and leukocyte counts were measured. In addition, kidneys, liver, spleen and lungs were investigated for fibrin deposition by immunohistochemistry and Western-blotting. Frozen sections were analysed for double labeling for platelet and leukocyte presence. RESULTS After coll/epi challenge, the number of platelets and leukocytes decreased significantly in both genotypes. Lower agonist concentration resulted in an attenuated platelet decrease in PSGL-1 knockout mice compared to the controls, however changes in leukocyte and neutrophil counts were not significantly different in the two strains. In knockout mice considerably less fibrin deposition has been observed in the lungs by Western-blotting and immunohistochemistry. After coll/epi challenge the lungs of the PSGL-1 knockout animals contained both platelets and leukocytes but less thrombi has been detected than in wild-type mice. CONCLUSIONS Our results indicate that the deficiency of PSGL-1 results in milder thrombocytopenia, less fibrin deposition and lower number of thrombosed blood vessels, suggesting that this molecule is essential for multicellular interactions during thrombus formation.

Application of a low molecular weight antifungal protein from Penicillium chrysogenum (PAF) to treat pulmonary aspergillosis in mice

PAF, a small antifungal protein from Penicillium chrysogenum, inhibits the growth of several pathogenic filamentous fungi, including members of the Aspergillus genus. PAF has been proven to have no toxic effects in vivo in mice by intranasal application. To test its efficacy against invasive pulmonary aspergillosis (IPA), experiments were carried out in mice suffering from IPA. Adult mice were immunosuppressed and then infected with Aspergillus fumigatus. After stable infection, the animals were inoculated with PAF intranasally at a concentration of 2.7 mg/kg twice per day. At this concentration—which is highly toxic in vitro to A. fumigatus—the mortality of the animals was slightly delayed but finally all animals died. Histological examinations revealed massive fungal infections in the lungs of both PAF-treated and untreated animal groups. Because intranasally administered PAF was unable to overcome IPA, modified and combined therapies were introduced. The intraperitoneal application of PAF in animals with IPA prolonged the survival of the animals only 1 day. Similar results were obtained with amphotericin B (AMB), with PAF and AMB being equally effective. Combined therapy with AMB and PAF—which are synergistic in vitro—was found to be more effective than either AMB or PAF treatment alone. As no toxic effects of PAF in mammals have been described thus far, and, moreover, there are so far no A. fumigatus strains with reported inherent or acquired PAF resistance, it is worth carrying out further studies to introduce PAF as a potential antifungal drug in human therapy. Emerging Microbes & Infections (2016) 5, e114; doi:10.1038/emi.2016.116; published online 9 November 2016

Interpretation of osmotic gradient ektacytometry (osmoscan) data: A comparative study for methodological standards

Abstract Osmotic gradient ektacytometry (measuring elongation index in the function of osmolality at a constant shear stress) is a sensitive method to analyze red blood cell (RBC) deformability and investigating the optimal osmolality range for the cells in normal or pathophysiological cellular and micro-environmental conditions. However, the methodological conditions are different, since the results are influenced by the applied shear stress (SS). In this study we investigated rat, dog, pig and human blood samples at SS of 1, 2, 3, 5, 10, 20 and 30 Pa. To describe the range being related to the cell deformability, we introduced new calculated parameters obtained from the raw data of the elongation index (EI)-osmolality (O) curves. Our results showed that: (1) Osmoscan data tested at 20 or 30 Pa do not differ significantly from each other; (2) Under SS of 20 Pa the EImax, the O (EImax), the EI min and the area under curve nearly linearly decrease in the function of SS with different slope in rat, dog, pig and human blood; (3) Measurements under 3 Pa SS become unstable; (4) The differences between minimal and maximal EI and the belonging osmolality values, and their ratios, as new calculated parameters (ΔEI, ΔO, ΔEI/ΔO, EImax/EImin and O (EImax)/Omin) can be suitable for further analysis of the osmoscan curves together with other hemorheological parameters describing RBC deformability; and (5) Decreased erythrocyte deformability (by rigidifying with glutaraldehyde) can be reflected well with the following, calculated osmoscan parameters: ΔO, rO, rEI/rO and ΔEI/ΔO.

Following-up hemorheological consequences of gonadectomy in male and female rats

Growing number of clinical and experimental data reflect to the gender differences of hemorheological parameters. However, little is known about the potential hemorheological effect of gonadectomy and consequent changes in sex hormone concentration. Adult, same-aged male and female rats were involved in the study. In control male and female group no surgical intervention was performed. In gonadectomized (GoE) male and female groups bilateral orchidectomy or ovariectomy were completed. Body weight measurement and blood sampling were carried out in the 1st, 2nd and 3rd postoperative months. The GoE females had significant bodyweight augmentation and their plasma estrogen concentration decreased by 40-45% by the 1st postoperative month, while in males the testosterone level was not detectable after gonadectomy. Leukocyte and platelet counts moderately increased in GoE males. Elongation index values of erythrocytes slightly decreased in both genders after gonadectomy, showing converging values. Erythrocyte aggregation index values of GoE females significantly raised by the 2nd month. It can be concluded that gonadectomy in rats resulted in alteration (dominantly impairment) of blood microrheological parameters, by different manner in males and females. Supposedly decrease in estrogen can cause more expressed hemorheological changes than the cessation of testosterone.

The effect of centrifugation at various g force levels on rheological properties of rat, dog, pig and human red blood cells

Laboratory investigations often require centrifugation of blood samples for various erythrocyte tests. Although there is a lack of data about the effect of centrifugation at various g force levels on erythrocyte rheological properties. We aimed to investigate the effect of a 10-minute centrifugation at 500, 1000 or 1500 g at 15°C of rat, dog, pig and human venous (K3-EDTA, 1.5 mg/ml) blood samples. Hematological parameters, erythrocyte deformability, cell membrane stability, osmotic gradient ektacytometry (osmoscan) and erythrocyte aggregation were determined. Hematological and erythrocyte deformability parameters showed interspecies differences, centrifugation caused no significant alterations. Cell membrane stability for human erythrocytes centrifuged at higher g level showed less decrease in deformability. Osmoscan O min parameter showed slight elevation in dog centrifuged aliquots. Erythrocyte aggregation parameters changed unexpectedly. Rat and dog erythrocyte aggregation indices significantly dropped in centrifuged aliquots. Pig erythrocyte aggregation indices increased significantly after centrifugation. Human erythrocyte aggregation was the most stable one among the investigated species. The used centrifugation protocols caused the largest alterations in erythrocyte aggregation in a controversial way among the investigated species. On the other hand, erythrocyte deformability parameters were stable, cell membrane stability and osmoscan data show minor shifts.

Micro-rheological changes during experimental acute pancreatitis in the rat

Although microcirculatory disturbances play pivotal role in the pathomechanism of acute pancreatitis (AP), very few papers can be found which had been tested any of hemorheological parameters. The aim of our study was to analyze the hemorheological changes in cerulein-induced experimental acute pancreatitis in rat in two doses (5 and 10 μg/kg, s.c.). Male and female rats were subjected to Control group, or AP with 5 or 10 μg/kg cerulein groups. Blood samplings (lateral caudal vein) were completed before cerulein administration, and 1, 2 and 24 hours later. Hematological parameters, amylase activity, erythrocyte deformability (ektacytometry) and aggregation (light-transmission method) were tested. The presence of AP could be confirmed by amylase testing and histological examination. The earliest impairment of the red blood cell deformability could be observed 1 hour after cerulein administration in 10 μg/kg dosage. Female animals had the worst rheological results with high mortality. In conclusion, subcutaneously administrated cerulein in dosage of 5 and 10 μg/kg resulted in AP in rats, with significant changes in red blood cell deformability and alterations in erythrocyte aggregation. This model seems to be suitable for further comparative studies.