Barbara Dołęgowska

An intensified systemic trafficking of bone marrow‐derived stem/progenitor cells in patients with pancreatic cancer

Various experimental studies indicate potential involvement of bone marrow (BM)‐derived stem cells (SCs) in malignancy development and progression. In this study, we comprehensively analysed systemic trafficking of various populations of BM‐derived SCs (BMSCs), i.e., mesenchymal, haematopoietic, endothelial stem/progenitor cells (MSCs, HSCs, EPCs respectively), and of recently discovered population of very small embryonic/epiblast‐like SCs (VSELs) in pancreatic cancer patients. Circulating CD133+/Lin−/CD45−/CD34+ cells enriched for HSCs, CD105+/STRO‐1+/CD45− cells enriched for MSCs, CD34+/KDR+/CD31+/CD45− cells enriched for EPCs and small CXCR4+CD34+CD133+ subsets of Lin−CD45− cells that correspond to VSELs were enumerated and sorted from blood samples derived from 29 patients with pancreatic cancer, and 19 healthy controls. In addition, plasma levels of stromal‐derived factor‐1 (SDF‐1), growth/inhibitory factors and sphingosine‐1‐phosphate (S1P; chemoattractants for SCs), as well as, of complement cascade (CC) molecules (C3a, C5a and C5b‐9/membrane attack complex – MAC) were measured. Higher numbers of circulating VSELs and MSCs were detected in pancreatic cancer patients (P < 0.05 and 0.01 respectively). This trafficking of BMSCs was associated with significantly elevated C5a (P < 0.05) and C5b‐9/MAC (P < 0.005) levels together with S1P concentrations detected in plasma of cancer patients, and seemed to be executed in a SDF‐1 independent manner. In conclusion, we demonstrated that in patients with pancreatic cancer, intensified peripheral trafficking of selected populations of BMSCs occurs. This phenomenon seems to correlate with systemic activation of the CC, hepatocyte growth factor and S1P levels. In contrast to previous studies, we demonstrate herein that systemic SDF‐1 levels do not seem to be linked with increased mobilization of stem cells in patients with pancreatic cancer.

Novel evidence that crosstalk between the complement, coagulation and fibrinolysis proteolytic cascades is involved in mobilization of hematopoietic stem/progenitor cells (HSPCs).

The role of blood proteinases in the mobilization of hematopoietic stem/progenitor cells (HSPCs) is still not well understood. As previously reported, activation of the complement cascade (ComC) and cleavage of C5 by C5 convertase are enabling events in the release of C5a that plays a crucial role in the egress of HSPCs from bone marrow (BM) into peripheral blood (PB) and explains why C5-deficient mice are poor mobilizers. Here we provide evidence that during granulocyte colony-stimulating factor- and AMD3100-induced mobilization, not only the ComC but also two other evolutionarily ancient proteolytic enzyme cascades, the coagulation cascade (CoaC) and the fibrynolytic cascade (FibC), become activated. Activation of all three cascades was measured by generation of C5a, decrease in prothrombin time and activated partial thromboplastin time as well as an increase in the concentrations of plasmin/antiplasmin and thrombin/antithrombin. More importantly, the CoaC and FibC, by generating thrombin and plasmin, respectively, provide C5 convertase activity, explaining why mobilization of HSPCs in C3-deficient mice, which do not generate ComC-generated C5a convertase, is not impaired. Our observations shed more light on how the CoaC and FibC modulate stem cell mobilization and may lead to the development of more efficient mobilization strategies in poor mobilizers. Furthermore, as it is known that all these cascades are activated in all the situations in which HSPCs are mobilized from BM into PB (for example, infections, tissue/organ damage or strenuous exercise) and show a circadian rhythm of activation, they must be involved in both stress-induced and circadian changes in HSPC trafficking in PB.

Conjugated linoleic acid increases intracellular ROS synthesis and oxygenation of arachidonic acid in macrophages

OBJECTIVE Conjugated linoleic acids (CLAs) have potential antiatherosclerotic properties: they may inhibit atherosclerotic processes by reducing the intensity of inflammatory processes. However, in vivo studies have shown that the application of trans-10, cis-12 CLA in obese men increased their oxidative stress. The objective of this study was to determine whether CLA can lead to an increase in oxidative stress and to isoprostane synthesis in macrophages. METHODS Monocytes from peripheral blood and human monocytic leukemia cells were used in this study. Monocytes were differentiated to macrophages, and were incubated with 30 microM cis-9, trans-11 CLA and trans-10, cis-12 CLA or linoleic acid for 2 days. In some experiments the inhibitors of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) or respiratory chain were added. After incubation, synthesis of reactive oxygen species (ROS), total cellular concentration of adenosine triphosphate, concentration of 8-epi-prostaglandin F2 alpha, activity of cytoplasolic phospholipase A2 (cPLA2), activity of mitochondria, and expression of mRNA of PPAR-alpha were measured. RESULTS In cells cultured with CLAs intercellular ROS synthesis increased. In this condition the mitochondrial energy potential was high, and the inhibitors of the respiratory chain and PPAR-alpha reduced ROS concentration. At the same time, the cPLA2 activity was abolished. In contrast, 8-iPF2 alpha III synthesis increased in CLA cells. CONCLUSION Cultivation of cells with CLA leads to an increased ROS synthesis, partly by PPAR-alpha mechanism. An increase in ROS concentration and inhibition of cPLA2 activity can stimulate oxygenation of arachidonic acid and contribute to an increase in 8-epi-PF2 alpha III level and in the apoptosis process in macrophages.

Relationship between the concentrations of heavy metals and bioelements in aging men with metabolic syndrome.

Heavy metals may exacerbate metabolic syndrome (MS) but abnormal serum concentrations of bioelements may also co-exist with MS. The primary aim of the study was to assess the relationship of blood heavy metal and bioelement concentrations and MS, in men aged 50–75 years. Heavy metals—lead (Pb), cadmium (Cd), mercury (Hg), arsenic (As), tungsten (W), Macroelements—magnesium (Mg) and calcium (Ca), and microelements—iron (Fe), zinc (Zn) copper (Cu), chromium (Cr), molybdenum (Mo), selenium (Se) and manganese (Mn), body mass index (BMI), waist to hip ratio (WHR), abdominal circumference (AC) and blood pressure (BP), total cholesterol (TCh), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride (TG), fasting plasma glucose (FPG), insulin, and Homeostasis Model Assessment—Insulin resistance (HOMA-IR). The men with MS showed statistically significant higher Zn and lower Mg concentrations. Those with diabetes had higher Ca concentration and lower Mg concentration. Cr and Mn concentrations were significantly higher in obese men. The participants with hypertension had lower Mg concentration. We found statistically significant positive correlations (W-TCh, W-LDL, Mg-TCh, Mg-LDL, Ca-TCh, Ca-LDL, Ca-insulin, Ca-HOMAR-IR, Zn-TG, Zn-insulin, Zn-HOMA-IR, Cu-BP systolic, Mn-BMI, Mn-AC, Mn-WHR, Mn-insulin, Mn-HOMA-IR, Se-TCh, Se-LDL, Se-TG, Se-insulin, Se-HOMA-IR, Cr-TCh, Cr-HDL, Cr-LDL, Cr-TG) and negative correlations (Cd-insulin, Hg-WHR, W-insulin, W-HOMA-IR, Mg-BMI, Mg-AC, Mg-WHR, Mg-BP systolic, Mo-insulin, Mn-HDL). Tungsten may contribute to lipid disorders. Magnesium appears to play the protective role in the occurrence of metabolic disorders. Microelements Mn, Cr and Se may intensify MS.

Whole-Body Cryostimulation - Potential Beneficial Treatment for Improving Antioxidant Capacity in Healthy Men - Significance of the Number of Sessions

It is claimed that WBC (whole-body cryotherapy) enhances the resistance of the human body, also thanks to the beneficial effect on the antioxidant system. Accordingly, this research aimed to evaluate the effect of a series of whole-body cryostimulations on the level of non-enzymatic antioxidants and the activity of antioxidant enzymes in healthy men. The study was carried out on 30 young and healthy men aged 27.8±6.1 years with average body mass index and peak oxygen consumption (46.34±6.15 ml kg−1 •min−1). The participants were daily exposed for 3 minutes to cryogenic temperatures (−130°C). Blood samples were obtained in the morning before cryostimulation, again 30 min after exposure and the following day in the morning, during the 1st, 10th and 20th session. Analysis concerned changes in plasma concentrations of total protein, albumin, glucose, uric acid and ceruloplasmin, and the most important components of the antioxidant system in red blood cells: superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, reduced and oxidized glutathione. To assess the oxidative stress level the 8-isoprostane concentration in plasma was measured. The obtained results indicate that cryogenic temperatures in repeated daily treatments result in changes in the peroxidant and antioxidant status. These changes seem to depend on the number of cryostimulations. After 20 daily treatments there was an increase in SOD, SOD:CAT ratio, a decrease in the concentration of reduced and oxidized glutathione and in the activity of GPx. It could be possible that differences in the activity of GSSG-R after 20 treatments depended on the body mass index of participants.

Clinical analysis of perioperative complement activity during ischemia/reperfusion injury following renal transplantation.

BACKGROUND AND OBJECTIVES The complement cascade seems to be an important mediator modulating renal ischemia/reperfusion injury. This study analyzed whether significant changes occur in the levels of a terminal panel of complement molecules (C3a, C5a, and C5b-9/membrane attack complex) during the early phase of human kidney allograft reperfusion and evaluated the potential association of these changes with clinical post-transplant graft function in kidney transplant recipients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Seventy-five renal transplant recipients undergoing transplantation between 2004 and 2006 were enrolled in the study and divided into early, slow, and delayed graft function groups. Blood samples were collected perioperatively during consecutive minutes of allograft reperfusion from the renal vein. Levels of complement molecules were measured using ELISA. RESULTS Analysis revealed no significant changes in C3a and C5a levels throughout reperfusion. The main complement molecule that was significantly associated with post-transplant graft function was C5b-9/membrane attack complex; throughout the reperfusion period, perioperative levels of C5b-9/membrane attack complex were around two to three times higher in delayed graft function patients than early and slow graft function individuals (P<0.005). In addition, C5b-9/membrane attack complex levels had a relatively high clinical sensitivity and specificity (70%-87.5%) for the prediction of early and long-term (1 year) post-transplant allograft function. CONCLUSIONS This clinical study supports a role for the complement cascade in delayed graft function development. However, additional studies are needed to elucidate the exact mechanisms responsible for this phenomenon. In addition, perioperative measurements of C5b-9/membrane attack complex are highlighted as promising potential clinical markers of post-transplant renal allograft function.

Activity of selected enzymes in erythrocytes and level of plasma antioxidants in response to single whole‐body cryostimulation in humans

The influence of extremely low temperatures on the human body and physiological reactions is not fully understood. The aim of this research was to estimate the influence of a single exposure to cryogenic temperature (−130°C), without subsequent kinesiotherapy, on the activity of the most crucial antioxidant enzymes in erythrocytes: superoxide dismutase (SOD), catalase (CAT), glutathione reductase (R‐GSSG), glutathione peroxidase (GPx) and glutathione transferase (T‐GSH). In the plasma, the concentrations of glutathione, uric acid, albumins and extra‐erythrocyte haemoglobin as components of the non‐enzymatic antioxidant system were evaluated. The subjects were 10 healthy young men. Blood was sampled in the morning on the day of cryostimulation, 30 min after cryostimulation and the next morning. The enzymatic response of the antioxidant defence to the influence of the extremely low temperature resulted in an immediate, significant, increase in GPx and R‐GSSG activities, but a decrease in CAT and T‐GSH activities. We observed an increase in the concentrations of all the examined non‐enzymatic antioxidants, especially extra‐erythrocyte haemoglobin and uric acid, which had both increased further the day after cryostimulation. The results indicate that a single stimulation with cryogenic temperatures results in oxidative stress in a healthy body, but that the level of stress is not very high. It seems that in this case the most significant role in the antioxidant mechanisms is played by peroxidase.

Selected Cytokines in Patients with Pancreatic Cancer: A Preliminary Report

Background/Aims Recent experimental studies have suggested that various cytokines may be important players in the development and progression of pancreatic cancer. However, these findings have not yet been verified in a clinical setting. Methods In this study, we examined the levels of a broad panel of cytokines, including interleukin (IL)-1, IL-6, IL-8, IL-10, IL-12, IL-17, and IL-23, as well as tumor necrosis factor alpha (TNFα) and granulocyte-colony stimulating factor (G-CSF) in patients with pancreatic adenocarcinoma (n = 43), other pancreatic malignancies (neuroendocrine [n = 10] and solid pseudopapillary tumors [n = 3]), and healthy individuals (n = 41). Results We found that there were higher levels of IL-6, IL-8, IL-10 and TNFα in patients with pancreatic cancer compared to healthy controls (for all, at least p<0.03). Cancer patients had lower IL-23 concentrations than healthy individuals and patients diagnosed with other types of malignancies (for both, p = 0.002). Levels of IL-6, IL-8, IL-10, and IL-23 were significantly associated with the direct number of circulating bone marrow (BM)-derived mesenchymal or very small embryonic/epiblast-like stem cells (SCs) in patients with pancreatic cancer. Moreover, our study identified a potential ability of IL-6, IL-8, IL-10, IL-23, and TNFα levels to enable discrimination of pancreatic cancer from other pancreatic tumors and diseases, including acute and chronic pancreatitis and post-pancreatitis cysts (with sensitivity and specificity ranging between 70%–82%). Conclusions Our study i) supports the significance of selected cytokines in the clinical presentation of pancreatic cancer, ii) highlights numerous associations between selected interleukins and intensified BMSCs trafficking in patients with pancreatic cancer, and iii) preliminarily characterizes the diagnostic potential of several cytokines as potential novel clinical markers of pancreatic cancer in humans.

Platelets arachidonic acid metabolism in patients with essential hypertension.

Arachidonic acids (AA) metabolites, eicosanoids, exert a tremendous influence on circulatory and vascular homeostasis, and in humans are generated by many organs and cell types. In this study we wanted to verify whether platelets AA metabolism play a significant role in pathogenesis of essential hypertension (EH). Participants were divided into the study (EH) and the control group. Plasma and urine concentrations of isoprostanes (8-iPF2α-III) and thromboxane B2 (TxB2) were determined using the ELISA method. The levels of 5- and 12-hydroxyeicosatetraenoic (HETE) acids, generated by platelets, were analysed using RP-HPLC. In a suspension of not stimulated and AA-stimulated platelets TxB2 level was statistically lower in the study than in the control group (p < 0.0001 and 0.001 respectively). The concentration of 12-HETE was significantly elevated in EH patients compared to the control group; however, only in the non-stimulated conditions (p < 0.05). Plasma and urine F2-isoprostanes levels were significantly higher in hypertensive individuals than in the control group (p < 0.00002 and p < 0.01 respectively). Moreover, EH patients excreted more TxB2 in urine than normotensive individuals (p < 0.05). Our results highlight the mutual connections between the platelets AA metabolism and indicate its possible role in the pathogenesis of arterial hypertension. Moreover, we hypothesize that platelets AA metabolism may exert a pro-atherosclerotic effect. Finally, we suggest the use of (5-HETE+12-HETE)/TxB2 parameter in further studies.

Oxidative Stress and Antioxidative Enzyme Activities in Chronic Kidney Disease and Different Types of Renal Replacement Therapy

The incidence and diagnosis of chronic kidney disease (CKD) is on the rise all over the world. CKD is related to ageing of the society and high morbidity due to lifestyle diseases like diabetes, atherosclerosis, and hypertension. CKD is associated with increased oxidative stress generated by uremic toxicity, chronic inflammatory state, lack of vitamins and microelements, parenteral iron administration, and dialysis procedure itself. In terms of cellular physiology, erythrocytes and blood platelets in particular have effective enzymatic and non-enzymatic antioxidative system. The most efficient enzymatic antioxidants include superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase. Glutathione is the leading non-enzymatic free radical scavenger. In CKD, antioxidative defense is impaired and the abnormal activity of the enzymes and glutathione concentration is described in literature. The imbalance between the formation of reactive oxygen species and antioxidative system efficiency takes part in the pathogenesis of cardiovascular complications. It contributes to increased morbidity and mortality in patients with CKD. The severity of these processes depends on the type of renal replacement therapy; haemodialysis (HD) is more predisposing to such disorders than peritoneal dialysis (PD), or even conservative treatment. This can influence the outcome and the possibility of kidney transplantation. Moreover, the early function of kidney allograft seems to be dependent on perioperative antioxidative ability of platelets, which can play a potential protective role in kidney transplantation.