Transplantation of mesenchymal stem cells cultured on biomatrix support induces repairing of digestive tract defects, in animal model.

Transplanted mesenchymal stem cells (MSCs) appear to play a significant role in adult tissue repair. The aim of this research was to obtain MSCs enriched, three dimensional (3D) patches for transplant, and to test their ability to induce repair of iatrogenic digestive tract defects in rats. MSCs were obtained from human and rat bone marrow, cultured in vitro, and seeded in a collagen-agarose scaffold, where they showed enhanced viability and proliferation. The phenotype of the cultured cells was representative for MSCs (CD105+, CD90+, and CD34-, CD45-, CD3-, CD14-). The 3D patch was obtained by laying the MSCs enriched collagen-agarose scaffold on a human or swine aortic fragment. After excision of small portions of the rat digestive tract, the 3D patches were sutured at the edge of the defect using micro-surgical techniques. The rats were sacrificed at time-points and the regeneration of the digestive wall was investigated by immunofluorescence, light and electron microscopy. The MSCs enriched 3D patches were biocompatible, biodegradable, and prompted the regeneration of the four layers of the stomach and intestine wall in rats. Human cells were identified in the rat regenerated digestive wall as a hallmark of the transplanted MSCs. For the first time we constructed 3D patches made of cultured bone marrow MSCs, embedded into a collagen-rich biomatrix, on vascular bio-material support, and transplanted them in order to repair iatrogenic digestive tract defects. The result was a complete repair with preservation of the four layered structure of the digestive wall.

Low basal levels of circulating adiponectin in patients undergoing coronary stenting predict in-stent restenosis, independently of basal levels of inflammatory markers: lipoprotein associated phospholipase A2, and myeloperoxidase.

OBJECTIVE: The aim of this study was to find a pre-interventional marker with the capacity to predict in-stent restenosis (ISR). Considering the anti-atherosclerotic role of adiponectin (APO), an adipocytokine with anti-inflammatory, anti-proliferative, anti-oxidative and anti-thrombotic properties, low plasma levels of APO might be correlated with the risk of ISR. We investigated the correlations between the plasma levels of APO and two markers of inflammation: lipoprotein associated phospholipase A2 (Lp-PLA2) and myeloperoxidase (MPO). DESIGN AND METHODS: 80 patients with angiographically significant stenosis underwent percutaneous coronary intervention (PCI) with bare metal stent. Plasma APO concentration and plasma Lp-PLA2 and MPO activities were evaluated immediately before and after PCI, then followed-up at 24, 48, 72 h, and at 1, 3, 6 months, respectively. ISR was evaluated at 6 months after stenting by follow-up coronary angiograms, and it was defined as >50% stenosis of the target lesion. RESULTS: ISR was present in 33.75% of patients. Baseline APO plasma concentration, measured before PCI, was lower in ISR patients than those without ISR [3.97 (+/-1.05) vs 6.65 (+/-2.95) microg/mL respectively, p<0.001]. The patients with APO values less than 4.9 microg/mL at discharge were more susceptible to develop ISR (odd ratio, 4.27; 95% CI, 1.56-11.72, p<0.001). ISR rate was independent of inflammation markers Lp-PLA2 and MPO baseline values, measured before PCI. CONCLUSIONS: The persistence of a low APO plasma level at discharge and 6 months afterwards may be used as a clinically useful marker for ISR prediction in patients undergoing PCI.

Imatinib inhibits spontaneous rhythmic contractions of human uterus and intestine.

The interstitial cells of Cajal (ICC) in the digestive tract and ICC-like cells in extradigestive organs express the c-kit tyrosine-kinase receptor, and have been implicated as pacemakers of smooth muscle spontaneous activity. We used imatinib mesylate (Glivec) to investigate whether c-kit activity of Cajal-like cells in human myometrium is involved in spontaneous rhythmic contractions of human uterine smooth muscle, taking intestinal smooth muscle as a reference tissue. We show that imatinib concentration-dependently inhibited the myogenic contractions of human myometrium in the organ bath, while it significantly affected noradrenaline or K(+)-induced contractions only at concentrations exceeding 50 muM. An inhibitory antibody directed against the extracellular domain of the platelet derived growth factor receptor (PDGFR), another target of imatinib that is expressed by the uterine muscle cells themselves, failed to affect myogenic contractions. These results suggest that Cajal-type cells of human myometrium, as well as ICC of intestinal smooth muscle, participate in myogenic contractile mechanisms, via a novel ligand-independent c-kit/CD117 tyrosine-kinase signaling.

[Expression of stem cell markers on fetal and tumoral human liver cells in primary culture]. / Expresia de markeri ai celulelor stem pe celulele în cultura primara izolate din ficatul fetal si ficatul tumoral uman.

Hepatic stem cells can be identified by the expression of putative markers such as CD117 (c-kit), CD90 (Thy-1), CD34, and HLA-DR. We have identified populations expressing these markers in both fetal and tumoral human liver by flow cytometry, using monoclonal antibodies against CD90, CD117, CD34, and HLA-DR. In tumoral liver CD117+/CD90+ cells were found in decreasing number from the neoplastic (2.48 +/- 0.67) and peritumoral region (0.88 +/- 0.12) to the area of para-tumoral (normal) parenchyma (0.13 +/- 0.04). The CD117+/CD34+ cells showed the following distribution: 0.35 +/- 0.05% in the tumoral region, 1.01 +/- 0.23% in the peritumoral region and 0.35 +/- 0.01 in the para-tumoral region. Using the same markers on fetal liver cells we have also identified small populations of CD117+/CD90+ cells (0.28 +/- 0.07%) and CD117+/CD34+ cells (1.13 +/- 0.24%), presumably resident stem cells or hematopoietic stem cells. Immunomagnetic negative separation was then performed on fetal liver cells using monoclonal antibodies against specific markers of hematopoietic lineages such as CD3, 14, 16, 19, 22, and CD56 to eliminate this population. The remaining cells were then incubated with fluorescently labeled monoclonal antibodies against CD90 and CD117 and analyzed using fluorescence microscopy. As expected these markers were expressed on the majority of the selected cells (89.28 +/- 9.56%). Isolation using appropriate markers and initiation of primary cultures is a first step to the therapeutic use of fetal stem cells and for the study of adult liver stem cells involvement in carcinogenesis.

Vesicle traffic through intercellular bridges in DU 145 human prostate cancer cells.

We detected cell-to-cell communication via intercellular bridges in DU 145 human prostate cancer cells by fluorescence microscopy. Since DU 145 cells have deficient gap junctions, intercellular bridges may have a prominent role in the transfer of chemical signals between these cells. In culture, DU 145 cells are contiguous over several cell diameters through filopodial extensions, and directly communicate with adjacent cells across intercellular bridges. These structures range from 100 nm to 5 microm in diameter, and from a few microns to at least 50-100 microm in length. Time-lapse imagery revealed that (1) filopodia rapidly move at a rate of microns per minute to contact neighboring cells and (2) intercellular bridges are conduits for transport of membrane vesicles (1-3 microm in diameter) between adjacent cells. Immunofluorescence detected alpha-tubulin in intercellular bridges and filopodia, indicative of microtubule bundles, greater than a micron in diameter. The functional meaning, interrelationship of these membrane extensions are discussed, along with the significance of these findings for other culture systems such as stem cells. Potential applications of this work include the development of anti-cancer therapies that target intercellular communication and controlling formation of cancer spheroids for drug testing.

Apoptotic rate in patients with myelodisplastic syndrome treated with modulatory compounds of pro-apoptotic cytokines.

Excessive apoptosis has a central role in ineffective hematopoiesis in myelodysplastic syndrome (MDS). The aim of the study was to quantify apoptosis and Bcl-2 expression in patients with MDS and to use these parameters in the evaluation of treatment efficacy with compounds modulating proapoptotic cytokines. Bone marrow (BM) samples from eight MDS patients were studied: four with refractory anemia and four with refractory anemia with ringed sideroblasts. Two patients with Hodgkin disease without BM determination were studied for control. Therapy consisted in administration of pentoxyphylline, dexamethasone and ciprofloxacin. Biochemical assay of apoptosis and Bcl-2 was performed using annexin V-biotin conjugate antibody and anti-human Bcl-2 antibody respectively, followed by streptavidine-peroxidase conjugate, and peroxidase substrate. Ultrastructural investigation of BM samples was performed with standard electron microscopy techniques. Most of BM hematopoietic cells in the MDS patients had ultrastructural features of various stages of apoptosis including chromatin condensation and margination, cytoplasm condensation and budding of nuclear and plasma membranes to produce apoptotic bodies. Bcl-2 expression showed an inverse correlation with the rate of the apoptotic process. Periodic evaluation of these two parameters has shown an increase of Bcl-2 expression and a decrease of apoptotic rate in patients who had responded to the treatment. Response to the treatment was appreciated in accordance with their transfusion needs. Treatment efficiency diminished in time. The rate of apoptosis was inversely correlated with the level of Bcl-2 expression. These results confirm the importance of the apoptotic process evaluation in monitoring MDS treatment.

Selective protection by phosphatidic acid against staurosporine-induced neuronal apoptosis.

Phosphatidic acid, the main product of lipid breakdown through phospholipase D activation, has been implicated in important signal transduction pathways able to influence cell fate in many ways. The purpose of this work was to determine possible effects of phosphatidic acid on neuronal cell death pathways. Here we used cerebellar granular cell cultures and cell death was triggered with either staurosporine or H(2)O(2). Cell viability was quantified by spectrophotometry, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test. Staurosporine (1-3 microM) or H(2)O(2) (50-800 microM) induced cell death in a dose-dependent manner. Using fluorescent staining (propidium iodide or annexin V-Cy3/6-carboxyfluorescein) we showed that cell death was mostly apoptotic in staurosporine treated cells and mostly non-apoptotic (necrotic) in H(2)O(2) treated cells. Phosphatidic acid was able to increase cell viability in staurosporine-, but not in H(2)O(2) - treated cells. We therefore conclude that phosphatidic acid has neuroprotective potential in neurons exposed to stimuli that trigger apoptosis.

Neoductular progenitor cells regenerate hepatocytes in severely damaged liver: a comparative ultrastructural study.

In severely injured liver, stem cells give rise to progeny that tend to replace lost hepatocytes. Neoductular reaction appears as an inherent stage of liver reconstruction following severe damage caused by different pathological mechanisms. Few ultrastructural types of progenitor cells have been described, and some molecular phenotypes of progenitor stages have been characterized, but the details of the differentiation process are largely unknown. We prepared for light and electron microscopy examination human liver from biopsies of patients with chronic active hepatitis, and rat liver with allyl alcohol-induced periportal necrosis. We found that progenitor neoductular cells acquire the hepatocytic polarity pattern during a multi-step process apparently involving cell migration and dissolution of neoductular basement membrane. An intermediate stage with "mixed" ductular and hepatocytic polarity was described.

Dantrolene protects neurons against kainic acid induced apoptosis in vitro and in vivo.

Apoptotic cell death induced by kainic acid (KA) in cultures of rat cerebellar granule cells (CGC) and in different brain regions of Wistar rat pups on postnatal day 21 (P21) was studied. In vitro, KA (100-500 microM) induced a concentration-dependent loss of cell viability in MTT assay and cell death had apoptotic morphology as studied by chromatin staining with propidium iodide (PI). In vivo, twenty-four hours after induction of status epilepticus (SE) by an intraperitoneal KA injection (5 mg/kg) we quantified apoptotic cells in hippocampus (CA1 and CA3), parietal cortex and cerebellum using PI staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique. We report that dantrolene, a specific ryanodine receptor antagonist, was able to significantly reduce the apoptotic cell death in CGC cultures and in hyppocampal CA1 and parietal cortex regions. Our finding can be valuable for neuroprotective therapy strategies in patients with repeated generalized seizures or status epilepticus.

Significance of platelet-activating factor acetylhydrolase in patients with non-insulin-dependent (type 2) diabetes mellitus.

BACKGROUND: Non-insulin dependent diabetes mellitus (NIDDM) represents an independent risk factor for cardiovascular diseases (CVD), being characterized by a continuous low-grade inflammation and endothelial activation state. Plasma platelet - activating factor - acetylhydrolases (PAF-AHs) are a subgroup of Ca(2+)-independent phospholipase A(2) family (also known as lipoprotein-associated phospholipases A(2)) that hydrolyze and inactivate the lipid mediator platelet-activating factor (PAF) and/or oxidized phospholipids. This enzyme is considered to play an important role in inflammatory diseases and atherosclerosis. The present study aims to investigate the relations between the levels of PAF-AH activity and LDL-cholesterol / HDL-cholesterol (LDL-ch / HDL-ch) ratio in NIDDM patients as compared to controls. METHODS: serum PAF-AH activity was measured in 50 patients with dyslipidemia, in 50 NIDDM patients and in 50 controls (normal lipid and glucose levels). Total cholesterol, LDL-ch, HDL-ch, triglyceride and blood glucose were determined in all subjects. RESULTS: All NIDDM patients display hiperlipidemia, with increased LDL-ch and triglyceride levels. There is a significant correlation between LDL-ch levels (especially LDL-ch / HDL-ch ratio) and PAF-AH activity in dyslipidemic and NIDDM patients. CONCLUSION: Diabetic and dyslipidemic patients have an increased plasma PAF-AH activity correlated with their LDL-ch levels and mainly with LDL-ch / HDL-ch ratio. Plasma PAF-AH high levels appear to be important as a risk marker for endothelial dysfunction in patients with NIDDM.

Messenger molecules of the phospholipase signaling system have dual effects on vascular smooth muscle contraction.

Background and methods. In order to investigate the role of phospholipases and their immediately derived messengers in agonist-induced contraction of portal vein smooth muscle, we used the addition in the organ bath of exogenous molecules such as: phospholipases C, A(2), and D, diacylglycerol, arachidonic acid, phosphatidic acid, choline. We also used substances modulating activity of downstream molecules like protein kinase C, phosphatidic acid phosphohydrolase, or cyclooxygenase. Results. a) Exogenous phospholipases C or A(2), respectively, induced small agonist-like contractions, while exogenous phospholipase D did not. Moreover, phospholipase D inhibited spontaneous contractions. However, when added during noradrenaline-induced plateau, phospholipase D shortly potentiated it. b) The protein kinase C activator, phorbol dibutyrate potentiated both the exogenous phospholipase C-induced contraction and the noradrenaline-induced plateau, while the protein kinase C inhibitor 1-(-5-isoquinolinesulfonyl)-2-methyl-piperazine relaxed the plateau. c) When added before noradrenaline, indomethacin inhibited both phasic and tonic contractions, but when added during the tonic contraction shortly potentiated it. Arachidonic acid strongly potentiated both spontaneous and noradrenaline-induced contractions, irrespective of the moment of its addition. d) In contrast, phosphatidic acid inhibited spontaneous contractile activity, nevertheless it was occasionally capable of inducing small contractions, and when repetitively added during the agonist-induced tonic contraction, produced short potentiations of the plateau. Pretreatment with propranolol inhibited noradrenaline-induced contractions and further addition of phosphatidic acid augmented this inhibition. Choline augmented the duration and amplitude of noradrenaline-induced tonic contraction and final contractile oscillations. Conclusions. These data suggest that messengers produced by phospholipase C and phospholipase A(2) contribute to achieve the onset and maintenance of contraction, while phospholipase D-yielded messengers appear to provide a delayed "on/off switch" that ultimately brings relaxation.