Zofia Sulowska

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.

Nitric oxide donors: Spermine/NO and diethylenetriamine/NO induce ovarian cancer cell death and affect STAT3 and AKT signaling proteins

The important features of cancer cells are uncontrolled growth and proliferation, as well as the ability to metastasis. These features depend mainly on the constant overexpression and activity of various cell signaling proteins, such as signal transducer and activator of transcription 3 (STAT3) and serine-threonine protein kinase AKT proteins. Nitric oxide (NO) has the potential of being anti-tumoral agent, however the exact character of anti-tumoral action of NO is still a matter of debate. In our research we used two NO donors, belonging to NONOates family, with different half-life times: spermine nitric oxide complex hydrate (SPER/NO t1/2=39min) and diethylenetriamine nitric oxide adduct (DETA/NO, t1/2=20h). We evaluated the cytotoxic effect of aforementioned NO donors on SK-OV-3 and OVCAR-3 ovarian cancer cell lines, as well as their effect on posttranslational modification of STAT3 and AKT proteins in these cells. We found that both NO donors present cytotoxic activity on the cancer cell lines, mainly through the induction of apoptosis. What is more, at the high concentration and longer exposure time they were also capable of inducing late apoptosis/necrosis. Both NO donors inhibited STAT3 and AKT3 proteins phosphorylation and down regulated their cytosolic levels, with DETA/NO being stronger inhibitor. We suggests, that NO donors have the potential to act as anti-tumoral agent through inhibiting cancer cell signaling and reducing their viability.

Cholesterol oxidase is indispensable in the pathogenesis of Mycobacterium tuberculosis.

Despite considerable research effort, the molecular mechanisms of Mycobacterium tuberculosis (Mtb) virulence remain unclear. Cholesterol oxidase (ChoD), an extracellular enzyme capable of converting cholesterol to its 3-keto-4-ene derivative, cholestenone, has been proposed to play a role in the virulence of Mtb. Here, we verified the hypothesis that ChoD is capable of modifying the bactericidal and pro-inflammatory activity of human macrophages. We also sought to determine the contribution of complement receptor 3 (CR3)- and Toll-like receptor 2 (TLR2)-mediated signaling pathways in the development of macrophage responses to Mtb. We found that intracellular replication of an Mtb mutant lacking a functional choD gene (ΔchoD) was less efficient in macrophages than that of the wild-type strain. Blocking CR3 and TLR2 with monoclonal antibodies enhanced survival of ΔchoD inside macrophages. We also showed that, in contrast to wild-type Mtb, the ΔchoD strain induced nitric oxide production in macrophages, an action that depended on the TLR2, but not the CR3, signaling pathway. Both wild-type and mutant strains inhibited the production of reactive oxygen species (ROS), but the ΔchoD strain did so to a significantly lesser extent. Blocking TLR2-mediated signaling abolished the inhibitory effect of wild-type Mtb on ROS production by macrophages. Wild-type Mtb, but not the ΔchoD strain, decreased phorbol myristate acetate-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are involved in both TLR2- and CR3-mediated signaling pathways. Our finding also revealed that the production of interleukin 10 by macrophages was significantly lower in ΔchoD-infected macrophages than in wild-type Mtb-infected macrophages. However, tumor necrosis factor-α production by macrophages was the same after infection with mutant or wild-type strains. In summary, we demonstrate here that ChoD is required for Mtb interference with the TLR2-mediated signaling pathway and subsequent intracellular growth and survival of the pathogen in human macrophages.

Spontaneous apoptosis of neutrophils in whole blood and its relation to apoptosis gene proteins.

Apoptosis of neutrophils limits their pro‐inflammatory potential. We tested the ability of fresh and cultured whole blood neutrophils to undergo spontaneous apoptosis and expression of p53, Fas/Apo‐1, bcl‐2 protein in the cells using flow cytometry. Neutrophil apoptosis was estimated using Annexin V and propidium iodide binding and verified under light microscopy. The percentage of early and late apoptotic neutrophils in the blood samples increased significantly after 20 h culture from 12.3 ± 14.2% and 4.3 ± 4.2% to 39.5 ± 14% and 15.3 ± 9.6%, respectively. The majority of late apoptotic neutrophils had altered morphology in FSC/SSC dot plot compared to alive or early apoptotic neutrophils. Cultured neutrophils presented markedly lower expression of bcl‐2 protein compared to fresh blood cells: 211 ± 321 median of fluorescence intensity (MFI) and 787 ± 1152 MFI, respectively. The increased percentage of late apoptotic cells after culture paralleled the increase in the Fas/Apo‐1 expression and negatively correlated with bcl‐2 expression. We noted intracellular expression of p53 protein in neutrophils, although the expression did not correlate neither to the percentage of the apoptotic neutrophils, nor to the Fas/Apo‐1 or bcl‐2 expression. Our results suggested that neutrophil apoptosis is gene regulated, moreover, we present a possibility to assess the neutrophil apoptosis and cellular expression of the proteins of apoptosis related genes in whole blood samples.

Production of Cytokines During Interaction of Peripheral Blood Mononuclear Cells with Autologous Ovarian Cancer Cells or Benign Ovarian Tumour Cells

Cytokines produced by tumour and immune cells may play a significant role in a modulation of immune cells response against tumour. We investigated an ability of peripheral blood mononuclear cells (PBMC) of patients with early and advanced stages of ovarian cancer and from non‐cancer patients to produce various cytokines in the presence or absence of autologous ovarian cancer (OC) cells or benign ovarian tumour (BOT) cells. Activated PBMC of patients with advanced stage of cancer produced slight amount of interferon γ (IFN‐γ) and what’s more, the production of IFN‐γ was decreased in the presence of OC cells. PBMC of patients with ovarian cancer or benign ovarian tumour generated comparable amounts of interleukin 6 and 10 (IL‐6, IL‐10), and transforming growth factor β1 (TGF‐β1). PBMC of the patients with cancer produced higher amount of tumour necrosis factor α (TNF‐α) than PBMC of non‐cancer patients. We demonstrated here that the reciprocal contact of OC cells from advanced cancer with autologous PBMC altered the direction of produced cytokines and leads to the down‐regulation of IFN‐γ and TNF‐α as well as to up‐regulation of immunosuppressive (IL‐10, TGF‐β1) and pro‐inflammatory (IL‐6) cytokines production.

The role of 3-ketosteroid 1(2)-dehydrogenase in the pathogenicity of Mycobacterium tuberculosis

BackgroundA growing body of evidence suggests that Mycobacterium tuberculosis (Mtb) uses the host’s cholesterol as a source of carbon and energy during infection. Strains defective in cholesterol transport or degradation exhibit attenuated growth in activated macrophages and diminished infectivity in animal models. The aim of this study was to evaluate intracellular replication of a cholesterol degradation-deficient Mtb mutant in human macrophages (MØ) in vitro and assess the functional responses of Mtb mutant-infected MØ.ResultsA mutant Mtb H37Rv strain containing an inactivated kstD gene (∆kstD), which encodes 3-ketosteroid 1(2)-dehydrogenase (KstD), was previously prepared using the homologous recombination-based gene-replacement technique. A control strain carrying the kstD gene complemented with an intact kstD was also previously constructed. In this study, human resting MØ were obtained after overnight differentiation of the human monocyte-macrophage cell line THP-1. Resting MØ were further activated with interferon-γ (IFN-γ). The ability of the kstD-defective Mtb mutant strain to replicate intracellularly in human MØ was evaluated using a colony-forming assay. Nitric oxide (NO) and reactive oxygen species (ROS) production by MØ infected with wild-type or ∆kstD strains was detected using Griess reagent and chemiluminescence methods, respectively. The production of tumor necrosis factor-α and interleukin-10 by MØ after infection with wild-type or mutant Mtb was examined using enzyme-linked immunosorbent assays.We found that replication of mutant Mtb was attenuated in resting MØ compared to the wild-type or complemented strains. Moreover, the mutant was unable to inhibit the NO and ROS production induced through Toll-like receptor 2 (TLR2) signaling in infected resting MØ. In contrast, mutant and wild-type Mtb behaved similarly in MØ activated with IFN-γ before and during infection.ConclusionsThe Mtb mutant ∆kstD strain, which is unable to use cholesterol as a source of carbon and energy, has a limited ability to multiply in resting MØ following infection, reflecting a failure of the ∆kstD strain to inhibit the TLR2-dependent bactericidal activity of resting MØ.

Influence of opioid peptides on human neutrophil apoptosis and activation in vitro

BACKGROUND: It has been shown that cells of the immune system release opioid peptides and possess receptors for them. The concentrations of opioid peptides in the peripheral circulation rapidly increase during inflammation and acute stress response. AIMS: The effect of opioid peptides Met-enkephalin (M-ENK) and beta-endorphin (beta-END) on the oxidative metabolism of normal human neutrophils and their death by apoptosis in vitro was investigated. METHODS: Isolated from peripheral blood, neutrophils were incubated in the presence or absence of 10(-6) to 10(-10) M of M-ENK and beta-END for 12 and 18 h. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V-FITC protein binding to the cell surface. The MTT-reduction assay was employed to estimate the oxidative metabolism of neutrophils. RESULTS: Treatment with M-ENK caused a significant increase in apoptotic cells after 18 h of culture: *0 M (control) versus 10(-10) M, p < or = 0.02; **10(-10) M versus 10(-10) M, p < or = 0.02. Treatment with beta-END caused a significant increase in apoptotic cells after 12 h of culture: 0 M versus 10(-8) M, p < or = 0.03; **0 M versus 10(-10) M, p < or = 0.04. We found the significant increase in MTT reduction by neutrophils in the presence of M-ENK and beta-END both before and after the culture. However, the ability of neutrophils to reduce the MTT salt to formazan decreased significantly after the culture. CONCLUSIONS: We observed that the in vitro effect of opioid peptides on the neutrophil survival and their functional state was time and dose dependent. The presence of antioxidants in the culture medium modifies neutrophil survival.

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.