Wojciech Błogowski

Various types of stem cells, including a population of very small embryonic-like stem cells, are mobilized into peripheral blood in patients with Crohn's disease†

Background: Developmentally early cells, including hematopoietic stem progenitor cells (HSPCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic‐like stem cells (VSELs), are mobilized into peripheral blood (PB) in response to tissue/organ injury. We sought to determine whether these cells are mobilized into PB in patients with Crohns disease (CD). Methods: Twenty‐five patients with active CD, 20 patients in clinical remission, and 25 age‐matched controls were recruited and PB samples harvested. The circulating CD133+/Lin−/CD45+ and CD34+/Lin−/CD45+ cells enriched for HSPCs, 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 the population of VSELs were counted by fluorescence‐activated cell sorting (FACS) and evaluated by direct immunofluorescence staining for pluripotency embryonic markers and by reverse‐transcription polymerase chain reaction (RT‐PCR) for expression of messenger (m)RNAs for a panel of genes expressed in intestine epithelial stem cells. The serum concentration of factors involved in stem cell trafficking, such as stromal derived factor‐1 (SDF‐1), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) were measured by enzyme‐linked immunosorbent assay (ELISA). Results: Our data indicate that cells expressing markers for MSCs, EPCs, and small Oct‐4+Nanog+SSEA‐4+CXCR4+lin−CD45− VSELs are mobilized into PB in CD. The mobilized cells also expressed at the mRNA level genes playing a role in development and regeneration of gastrointestinal epithelium. All these changes were accompanied by increased serum concentrations of VEGF and HGF. Conclusions: CD triggers the mobilization of MSCs, EPCs, and VSELs, while the significance and precise role of these mobilized cells in repair of damaged intestine requires further study. (Inflamm Bowel Dis 2012;)

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.

Identification of Very Small Embryonic/Epiblast-Like Stem Cells (VSELs) Circulating in Peripheral Blood During Organ/Tissue Injuries

We have identified in adult tissues a population of pluripotent very small embryonic/epiblast-like stem cells (VSELs) that we hypothesize are deposited at onset of gastrulation in developing tissues and play an important role as backup population of tissue-specific/committed stem cells. We envision that during steady-state conditions these cells may be involved in tissue rejuvenation and in processes of regeneration/repair after organ injuries. VSELs similarly as epiblast-derived migrating primordial germ cells change the epigenetic signature of some of the imprinted genes and therefore remain quiescent in adult tissues. These epigenetic changes in methylation status of imprinted genes prevent them also from teratoma formation. Mounting evidence indicates that VSELs are mobilized into peripheral blood during tissue/organ injuries and enumeration of these cells may be of prognostic value (e.g., in stroke or heart infarct). In this chapter, we will present FACS-based strategies to detect and enumerate these cells in human peripheral blood and umbilical cord blood.

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.

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.

Association between the perioperative antioxidative ability of platelets and early post-transplant function of kidney allografts: a pilot study.

Background Recent studies have demonstrated that the actions of platelets may unfavorably influence post-transplant function of organ allografts. In this study, the association between post-transplant graft function and the perioperative activity of platelet antioxidants was examined among kidney recipients divided into early (EGF), slow (SGF), and delayed graft function (DGF) groups. Methodology/Principal Findings Activities of superoxide dismutase, catalase, glutathione transferase (GST), glutathione peroxidase, and glucose-6-phosphate dehydrogenase (G6P) were determined and levels of glutathione, oxidized glutathione, and isoprostane were measured in blood samples collected immediately before and during the first and fifth minutes of renal allograft reperfusion. Our results demonstrated a significant increase in isoprostane levels in all groups. Interestingly, in DGF patients, significantly lower levels of perioperative activity of catalase (p<0.02) and GST (p<0.02) were observed. Moreover, in our study, the activity of platelet antioxidants was associated with intensity of perioperative oxidative stress. For discriminating SGF/DGF from EGF, sensitivity, specificity, and positive and negative predictive values of platelet antioxidants were 81–91%, 50–58%, 32–37%, and 90–90.5%, respectively. Conclusions During renal transplantation, significant changes occur in the activity of platelet antioxidants. These changes seem to be associated with post-transplant graft function and can be potentially used to differentiate between EGF and SGF/DGF. To the best of our knowledge, this is the first study to reveal the potential protective role of platelets in the human transplantation setting.

Adipose tissue as a potential source of hematopoietic stem/progenitor cells.

It has been more than 30 years since adipose tissue (AT) has been recognized as a central modulator orchestrating sophisticated process termed “immunometabolism”. Nonetheless, despite its unique involvement in the regulation of immune and endocrine homeostasis, recent studies demonstrated that AT also contains significant number of hematopoietic stem/progenitor cells (HSPCs) that may be there “settling down” throughout life. In this article we will focus on presenting the current concepts regarding endocrine, immunological, and molecular mechanisms that may contribute to and regulate bone marrow (BM)‐derived HSPCs homing into AT environment, as well as, highlight various structural and morphological similarities between BM and AT that might be involved in creating appropriate tissue niches for BM‐derived HSPCs in AT. Finally, we will discuss how development of obesity or type 2 diabetes may influence balance of homing signals for HSPCs in AT environment.

Is it possible to predict the early post-transplant allograft function using 20-HETE measurements? A preliminary report

20‐HydroxyEicosaTetraEnoic (20‐HETE) acid is an arachidonic acid metabolite that is generated via cytochrome P450 enzymes, and according to the findings from recent studies, may be involved in the pathogenesis of ischemia–reperfusion injury. The aim of this study was to: examine the dynamics of 20‐HETE changes during the first 5 min of allograft reperfusion, and analyze whether the observed changes are associated with post‐transplant graft function. Sixty‐nine renal transplant recipients were divided, according to their outcome, into early, slow and delayed graft function (EGF, SGF, DGF) group. Blood samples were collected directly before and during the first 5 min of allograft reperfusion. 20‐HETE concentrations were measured using ELISA. The results demonstrated significant differences in the concentrations and in the dynamics of 20‐HETE changes between patients with immediate graft function, and individuals with allograft activation problems. The sensitivity, specificity, positive and negative predictive value of 20‐HETEΔ(5‐0) parameter in discriminating EGF and SGF from DGF were 69%, 54%, 74% and 48% respectively. Therefore, our results demonstrated that the dynamics of 20‐HETE changes, which occurs during early phase of allograft reperfusion, is associated with early post‐transplant graft function and also highlighted 20‐HETE as a novel clinical marker of post‐transplant allograft function.

Clinical analysis of selected complement-derived molecules in human adipose tissue

BackgroundIt has been suggested that action of complement cascade [CC]-derived anaphylatoxins/molecules may represent a missing link between obesity and metabolic disorders. However, to date, the direct biochemical/immunomodulatory composition of the human AT environment remains poorly understood. In this study, we examined plasma and AT (subcutaneous and visceral/omental) levels of selected CC-derived anaphylatoxins/molecules, and adipsin as well as verified their associations with immune and stem cells chemoattractant - stromal-derived factor-1 (SDF-1).MethodsA total of 70 (35 subcutaneous and 35 omental) AT samples were obtained from patients undergoing elective surgery. Plasma and AT-derived interstitial fluid levels of C3a, C5a, C5b-9/membrane attack complex (MAC), complement factor D (adipsin) were measured using ELISA.ResultsAT levels of all examined substances were significantly lower than the corresponding levels in the plasma (in all cases P < 0.0000001). Moreover, in subcutaneous AT, robust C3a and adipsin concentrations were observed, whereas high levels of C5b-9/MAC were detected in the visceral depots. In addition, we established the correlations between analyzed molecular substances and body composition, BMI and/or the adiposity index of the examined patients.ConclusionsOur study demonstrated for the first time that significantly reduced levels of complement-derived molecules were present in human AT than in the peripheral blood, and that these factors are associated with the metabolic status of examined individuals. Moreover, in human AT, various associations between complement-derived molecules and SDF-1 levels exist.