Katarzyna Bednarska

Adenosine and 2'-deoxyadenosine modified with boron cluster pharmacophores as new classes of human blood platelet function modulators.

Novel types of adenosine and 2′‐deoxyadenosine derivatives containing boron clusters at positions C2′, N6, or C8 were synthesized. The effect of these modified compounds on platelet function was studied. Modification of adenosine at the C2′ position with a para‐carborane cluster (C2B10H11) results in efficient inhibition of platelet function, including aggregation, protein secretion, and P‐selectin expression induced by thrombin or ADP. These preliminary findings and the new chemistry proposed form the basis for the development of a new class of adenosine analogues that modulate human blood platelet activities.

Ovarian cancer cells modulate human blood neutrophils response to activation in vitro.

In cancer, numerous cells of both innate and adaptive immune systems are activated. Polymorphonuclear neutrophils are potent effector cells of inflammation that are an important component of tumour development and progression. The important signalling proteins that are involved in neutrophil functions are extracellular signal‐regulated kinases 1/2 (ERK1/2). We investigated the reactive oxygen species (ROS) production, adhesive ability and CD11b/CD18 adhesion molecule expression on neutrophils isolated from peripheral blood of ovarian cancer patients and the in vitro response of these cells to stimuli and direct contact with ovarian cancer cells isolated from tumour. We found that functional activities of neutrophils isolated from patients with advanced stages of ovarian cancer (FIGO III/IV) were intensified in comparison to neutrophils isolated from healthy female volunteers. Neutrophils of cancer patients produce higher amounts of ROS in response to stimuli than those of control group. Unstimulated neutrophils of patients possess higher expression of CD11b/CD18 molecule that is accompanied by increased adhesive ability of these cells. Our results reveal that augmented functional activities of neutrophils may result from the intensification of ERK1/2 kinases phosphorylation. We found that interactions with ovarian cancer cells modulate neutrophil functions as a result of cell‐to‐cell direct contact. We conclude that ovarian cancer cells affect pro‐inflammatory activities in neutrophils via influence of signalling pathways in response to stimuli. Our results suggest the possibility that neutrophils responding to contact with cancer cells contribute to the progression and metastatic potential of tumour cells.

Structure and serological characterization of the O-antigen of Proteus mirabilis O18 with a phosphocholine-containing oligosaccharide phosphate repeating unit.

A phosphorylated, choline-containing polysaccharide was obtained by O-deacylation of the lipopolysaccharide (LPS) of Proteus mirabilis O18 by treatment with aqueous 12% ammonia, whereas hydrolysis with dilute acetic acid resulted in depolymerisation of the polysaccharide chain by the glycosyl phosphate linkage. Treatment of the O-deacylated LPS with aqueous 48% hydrofluoric acid cleaved the glycosyl phosphate group but, unexpectedly, did not affect the choline phosphate group. The polysaccharide and the derived oligosaccharides were studied by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HMQC and HMQC-TOSCY experiments, along with chemical methods, and the following structure of the pentasaccharide phosphate repeating unit was established: [carbohydrate structure in text] Where ChoP=Phosphocoline Immunochemical studies of the LPS, O-deacylated LPS and partially dephosphorylated pentasaccharide using rabbit polyclonal anti-P. mirabilis O18 serum showed the importance of the glycosyl phosphate group in manifesting the serological specificity of the O18-antigen.

Seasonal changes in activities of human neutrophils in vitro

Objective and designWe present a retrospective analysis of previously collected blood samples to determine whether the immune response of neutrophils depends on the season i.e., short versus long days, in which blood samples were collected.MethodsThe bactericidal activity and adhesive capacity of neutrophils, the production of reactive oxygen species (ROS), and CD11b/CD18 molecule expression level were investigated. The investigated neutrophils were divided into two groups based on the time of blood collection: the winter season with short days and the summer season with long days.ResultsWe found seasonal variation in measurements of all the analyzed functional responses of neutrophils to stimuli. The strongest adhesion, as well as maximum values of ROS production, was presented by neutrophils isolated from the summer group. The highest bactericidal activity of neutrophils was also observed in blood donors from summer group.ConclusionsThe magnitude of the immune functional activity of neutrophils varies with the season of the year and is decreased in winter.

The interaction of HspA1A with TLR2 and TLR4 in the response of neutrophils induced by ovarian cancer cells in vitro

Inducible heat shock protein (HspA1A) promotes tumor cell growth and survival. It also interacts with effector cells of the innate immune system and affects their activity. Recently, we showed that the direct contact of ovarian cancer cells, isolated from tumor specimens, with neutrophils intensified their biological functions. Our current experiments demonstrate that the activation of neutrophils, followed by an increased production of reactive oxygen species, by cancer cells involves the interaction of HspA1A from cancer cells with Toll-like receptors 2 and 4 expressed on the neutrophils’ surface. Our data may have a practical implication for targeted anticancer therapies based, among other factors, on the inhibition of HspA1A expression in the cancer cells.

Signal transduction pathways affected by nitric oxide donors during neutrophil functional response in vitro.

Abstract.Objective and design:We investigated the intracellular signalling pathways by which nitric oxide (NO) donors: diethylamine/NO (DEA/NO) and 3-morpholinosydnonimine (SIN-1) regulate the functional response of human neutrophils to activating stimuli.Methods:The phosphorylation and nitration of signalling proteins, cyclic GMP level, neutrophil respiratory burst and adhesive activities and CD11b/CD18 molecule expression on neutrophils in the presence and absence of soluble guanylate cyclase inhibitors were determined.Results:NO donors showed strong inhibitory effect on activated neutrophils. NO donors nitrated the tyrosine residues in signalling proteins causing a decrease in tyrosine phosphorylation and neutrophils response to activation. Diethylamine/NO employed cyclic GMP as a signalling molecule in its action on neutrophils, whereas peroxynitrite anion donor affected neutrophil functions in a cGMP-independent manner. Moreover, we observed that peroxynitrite anion can overcome the nitric oxide molecule action.Conclusions:We conclude that each NO donor depending on its concentration and chemical nature may act on different elements of neutrophil signalling pathways capable of inducing distinct neutrophil functions.

Nitric oxide donors reduce the invasion ability of ovarian cancer cells in vitro

The most important factors involved in tumor metastasis and angiogenesis are metalloproteinases (MMPs), vascular endothelial growth factor, and multifunctional transforming growth factor &bgr;1. These factors are responsible for extracellular matrix degradation, induction of vascular permeability, and enhancement of tumor cells’ invasion and metastasis. Elevated expression and secretion of the above-mentioned factors are correlated with the higher aggressiveness of tumors and low patient survival for example, patients with ovarian cancer. Therefore, regulation of the expression, secretion, and activity of these factors is still considered a potent target for therapeutic intervention in cancer patients. Nitric oxide (NO) donors belong to the class of agents with multivalent targeted activities in cancer cells and are considered potential anticancer therapeutics. Our studies have shown that NO donors such as spermine/NO and diethylenetriamine/NO decrease the secretion of vascular endothelial growth factor-A from the OVCAR-3 ovarian cancer cell line, but not from the SK-OV-3 ovarian cancer cell line. The release of MMP-2 from both cell lines was reduced in a soluble guanylate cyclase-dependent manner by spermine/NO and diethylenetriamine/NO. Nevertheless, MMP-2 activity was only affected in SK-OV-3 cells. Both NO donors reduced the transmigration of the ovarian cancer cell lines. We did not observe any significant effect of spermine/NO and diethylenetriamine/NO on mRNA expression of the tested aggressiveness factors. In conclusion, our data indicated that NO donors reduced the metastatic potential of ovarian cancer cells, but its impact is rather low and requires high concentrations of donors. Moreover, both the tested cell lines differed in the susceptibility to NO donors.

Effect of nitric oxide donors on NADPH oxidase signaling pathway in human neutrophils in vitro

The production of reactive oxygen species (ROS) in activated neutrophils is catalyzed by NADPH oxidase, a multiproteins complex. Various protein kinases including protein kinase C (PKC) and mitogen activated protein kinases (MAPKs) are involved in NADPH oxidase phosphorylation and activation. The main step in the NADPH oxidase activation is phosphorylation of its cytosolic protein p47(phox). We found previously that nitric oxide (NO) donors such as metabolite of molsidomine-SIN-1 and diethylamine/NO significantly impaired the ROS production in activated human neutrophils. In this study, we investigated the effects of both NO donors on NADPH oxidase-linked signaling proteins in activated neutrophils. We found that NO donors decreased the phosphorylation of p47(phox) on tyrosine and serine/threonine residues and PKC on serine residues in neutrophils. Both NO donors did not affect the phosphorylation of MAPKs. NO donors partially but significantly lost their ability to reduce ROS production in the presence of PKC but not MAPKs inhibitors. We show that whereas NO donors have no effect on MAPKs activity, they do decrease PMA- and/or fMLP-induced phosphorylation of p47(phox) and PKC as well as PMA- and fMLP-induced ROS production.

Application of Intracellular Alkaline Phosphatase Activity Measurement in Detection of Neutrophil Adherence In Vitro

We have proposed the use of the fluorimetric method with 4-methylumbelliferyl phosphate (4-MUP) specific substrate for the alkaline phosphatase determination in the neutrophil adhesion assay. We provide evidence that the endogenous neutrophil alkaline phosphatase (NAP) activity evaluation is reliable to quantify neutrophil adhesion at a wide range of cell numbers (104−106). The results obtained by fluorimetric NAP activity test correlate to the results of adherence evaluated using the MTT reduction assay. The fluorimetric NAP activity test may be applied for resting as well as activated neutrophils without the risk of the activators interferences into the test. The alkaline phosphatase survey with the use of 4-MUP substrate is recommended herein as a sensitive, repeatable, simple, and reliable method of the neutrophil adherence determination in vitro.

JAK3, STAT3 and CD3-zeta signaling proteins status in regard to the lymphocytes function in patients with ovarian cancer.

Several groups of author have published that, in most cases of carcinoma, circulating lymphocytes are unable to carry out immune functions successfully. A molecular mechanism responsible for T lymphocytes defective reactivity in cancer patients is not completely defined. We evaluated whether the impaired function of peripheral blood lymphocytes (PBLs) from ovarian cancer patients could be associated with signaling elements such as JAK3, STAT3 and CD3-zeta chain. The study addressed to the simultaneous expression and phosphorylation status of mentioned molecules evaluation in regard to lymphocyte function in patients with advanced ovarian cancer has not yet been demonstrated by others. We found that PBLs of cancer patients showed lower JAK3, CD3-zeta molecules expression levels, as well as lower STAT3 and CD3-zeta phosphorylation levels than cells of control. The lower proliferative response and IL-2 production capacity of cancer patients PBLs in comparison with that of the control group cells were the functional consequences of reported in this study signaling abnormalities.