Abstract 1412: Sonic Hedgehog Gene Transfer Promotes Angiogenesis and Myocardial Regional Blood Flow via PKC Dependent Netrin Expression

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Rafeeq P Ahmed ◽  
Husnain K Haider ◽  
Shujia Jiang ◽  
Muhammed R Afzal ◽  
Muhammed Ashraf
Keyword(s):  
Blood Flow ◽  
Stem Cell ◽  
Gene Delivery ◽  
Conditioned Medium ◽  
Real Time ◽  
Real Time Pcr ◽  
Regional Blood Flow ◽  
Control Groups ◽  
Group 3 ◽  
Group 2

Background: Hedgehog signaling effects endothelial and fibroblast cell migration and vascularization of various tissues during embryonic development. We propose here that stem cell based Shh gene delivery to the heart promotes neovascularization and enhances regional blood flow (RBF) in the infartced heart. Methods and Results: The pCMVShh plasmid was constructed and used for non-viral transfection of primary mesenchymal stem cell (MSC) culture. Successful transfection and expression of Shh in transfected MSCs ( Shh MSCs) was confirmed by immunoflourescence, real-time PCR and Western blotting. Real-time PCR based array showed upregulation of angiogenic factors in Shh MSCs including netrin (>62-fold) and iNOS (>100-fold). The expression of netrin and iNOS was PKC dependent and was abolished in the presence of 2.5 μ M chelerythrin. Transduction of iNOS gene into MSCs increased netrin expression implying a role for iNOS downstream of PKC. Western blot showed that 45-KDa PKM proteolytic subunit of PKC was involved in this activity. Shh MSCs conditioned medium was biologically active and caused greater migration and tube formation of human vascular endothelial in vitro in comparison with untransfected MSCs ( non-Shh MSCs) conditioned medium. For in vivo studies, 70 μ l DMEM without cells (group-1) or containing 1x10 6 male non-Shh MSC (group-2), or Shh MSCs (group-3) were transplanted into female rat model of acute coronary artery ligation. S ry -gene studies on day-4 after cell engraftment showed higher survival of Shh MSCs ( p < 0.05 vs non-Shh MSC). Immunostaining for vWillebrand Factor-VIII and smooth muscle actin showed robust angiogenesis with distinctly large vessels in group-3 ( p < 0.001 vs group-2). RBF measured by fluorescent microspheres was increased ( p < 0.01 ) and infarction size was attenuated ( p < 0.05 ) in group-3 as compared with control groups. Left ventricle ejection fraction (52.3±2.6%) and fractional shortening (21.8±1.2%) were more preserved in group-3 as compared with control groups. Conclusions: Ex-vivo Shh gene delivery improves angiogenesis and RBF in the infarcted heart to preserve global heart function through the upregulation of multiple angiogenic cytokines including iNOS and netrin.

Correlation of γ-radioactivity and Scanning Electron Microscopy in measurement of retention of 111Indium-labeled canine endothelial cells on synthetic vascular grafts

1989 ◽  
Vol 47 ◽  
pp. 886-887
Author(s):  
E.J. Prendiville ◽  
S. Laliberté Verdon ◽  
K. E. Gould ◽  
K. Ramberg ◽  
R. J. Connolly ◽  
...  
Keyword(s):  
Blood Flow ◽  
Ex Vivo ◽  
Vascular Grafts ◽  
Retention Data ◽  
Vascular Prostheses ◽  
Group 4 ◽  
Human Fibronectin ◽  
Insufficient Time ◽  
Group 3 ◽  
Group 2

Endothelial cell (EC) seeding is postulated as a mechanism of improving patency in small caliber vascular grafts. However the majority of seeded EC are lost within 24 hours of restoration of blood flow in previous canine studies . We postulate that the cells have insufficient time to fully develop their attachment to the graft surface prior to exposure to hemodynamic stress. We allowed EC to incubate on fibronectin-coated ePTFE grafts for four different time periods after seeding and measured EC retention after perfusion in a canine ex vivo shunt circuit.Autologous canine EC, were enzymatically harvested, grown to confluence, and labeled with 30 μCi 111 Indium-oxine/80 cm 2 flask. Four groups of 5 cm x 4 mm ID ePTFE vascular prostheses were coated with 1.5 μg/cm.2 human fibronectin, and seeded with 1.5 x 105 EC/ cm.2. After seeding grafts in Group 1 were incubated in complete growth medium for 90 minutes, Group 2 were incubated for 24 hours, Group 3 for 72 hours and Group 4 for 6 days. Grafts were then placed in the canine ex vivo circuit, constructed between femoral artery and vein, and subjected to blood flow of 75 ml per minute for 6 hours. Continuous counting of γ-activity was made possible by placing the seeded graft inside the γ-counter detection crystal for the duration of perfusion. EC retention data after 30 minutes, 2 hours and 6 hours of flow are shown in the table.

scholarly journals Detection of β-Herpesviruses in Polish Adult Cord Blood Stem Cell Recipients by Real-Time PCR: Single Centre Study

2010 ◽  
Vol 58 (6) ◽  
pp. 467-472
Author(s):  
Tomasz Dzieciątkowski ◽  
Maciej Przybylski ◽  
Grzegorz Władysław Basak ◽  
Tigran Torosian ◽  
Agnieszka Tomaszewska ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Real Time ◽  
Real Time Pcr ◽  
Blood Stem Cell ◽  
Single Centre ◽  
Centre Study ◽  
Cord Blood Stem Cell ◽  
Single Centre Study

scholarly journals Implementation and Validation of the Roche Light Cycler 480 96-Well Plate Platform as a Real-Time PCR Assay for the Quantitative Detection of Cytomegalovirus (CMV) in Clinical Specimens Using the Luminex MultiCode ASRs System

2020 ◽  
Vol 8 (1) ◽  
pp. 14
Author(s):  
Shengwen Calvin Li ◽  
Kara J. Sparks ◽  
Leonard S. Sender
Keyword(s):  
Stem Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Range ◽  
Measurement Range ◽  
Quantitative Detection ◽  
Clinical Settings ◽  
Stem Cell Therapies ◽  
Cell Therapies ◽  
Copy Numbers

Allogenic stem-cell therapies benefit patients in the treatment of multiple diseases; however, the side effects of stem-cell therapies (SCT) derived from the concomitant use of immune suppression agents often include triggering infection diseases. Thus, analysis is required to improve the detection of pathogen infections in SCT. We develop a polymerase chain reaction (PCR)-based methodology for the qualitative real-time DNA detection of cytomegalovirus (CMV), with reference to herpes simplex virus types 1 (HSVI), Epstein–Barr virus (EBV), and varicella-zoster virus (VZV) in blood, urine, solid tissues, and cerebrospinal fluid. This real-time PCR of 96-well plate format provides a rapid framework as required by the Food and Drug Administration (FDA) for clinical settings, including the processing of specimens, reagent handling, special safety precautions, quality control criteria and analytical accuracy, precisely reportable range (analyst measurement range), reference range, limit of detection (LOD), analytical specificity established by interference study, and analyte stability. Specifically, we determined the reportable range (analyst measurement range) with the following criteria: CMV copies ≥200 copies/mL; report copy/mL value; CMV copies ≤199 copies/mL; report detected but below quantitative range; CMV copies = 0 with report <200 copies/mL. That is, with reference range, copy numbers (CN) per milliliter (mL) of the LOD were determined by standard curves that correlated Ct value and calibrated standard DNA panels. The three repeats determined that the measuring range was 1E2~1E6 copies/mL. The standard curves show the slopes were within the range −2.99 to −3.65 with R2 ≥ 0.98. High copy (HC) controls were within 0.17–0.18 log differences of DNA copy numbers; (2) low copy (LC) controls were within 0.17–0.18 log differences; (3) LOD was within 0.14–0.15 log differences. As such, we set up a fast, simple, inexpensive, sensitive, and reliable molecular approach for the qualitative detection of CMV pathogens. Conclusion: This real-time PCR of the 96-well plate format provides a rapid framework as required by the FDA for clinical settings.

Influence of adenosine on cerebral blood flow during hypoxic hypoxia in the newborn piglet

1990 ◽  
Vol 68 (4) ◽  
pp. 1534-1541 ◽  
Author(s):  
N. Laudignon ◽  
E. Farri ◽  
K. Beharry ◽  
J. Rex ◽  
J. V. Aranda
Keyword(s):  
Blood Flow ◽  
Cerebral Blood Flow ◽  
High Performance ◽  
Severe Hypoxia ◽  
Group 4 ◽  
Csf Levels ◽  
Group 3 ◽  
Group 2 ◽  
Arterial Po2 ◽  
Group 1

This study investigated the role of adenosine in the regulation of neonatal cerebral blood flow (CBF) during moderate (arterial PO2 = 47 +/- 9 Torr) and severe (arterial PO2 = 25 +/- 4 Torr) hypoxia. Twenty-eight anesthetized and ventilated newborn piglets were assigned to four groups: 8 were injected intravenously with the vehicle (controls, group 1); 13 received an intravenous injection of 8-phenyltheophylline (8-PT), a potent adenosine receptor blocker, either 4 mg/kg (group 2, n = 6, mean cerebrospinal fluid (CSF) levels less than 1 mg/l) or 8 mg/kg (group 3, n = 7, mean CSF levels less than 3.5 mg/l); and 7 received an intracerebroventricular injection of 10 micrograms 8-PT (group 4). During normoxia, CBF was not altered by vehicle or 8-PT injections. In group 1, 10 min of moderate and severe hypoxia increased total CBF by 112 +/- 36 and 176 +/- 28% (SE), respectively. Compared with controls, the cerebral hyperemia during moderate hypoxia was not altered in group 2, attenuated in group 3 (to 53 +/- 13%, P = NS), and completely blocked in group 4 (P less than 0.01). CBF increase secondary to severe hypoxia was attenuated only in group 4 (74 +/- 29%, P less than 0.05). CSF concentrations of adenosine and adenosine metabolites measured by high-performance liquid chromatography increased during hypoxia. Arterial O2 content was inversely correlated (P less than 0.005) to maximal CSF levels of adenosine (r = 0.73), inosine (r = 0.87), and hypoxanthine (r = 0.80).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Inhibition of Corneal Neovascularization by Topical and Subconjunctival Tigecycline

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Sertan Goktas ◽  
Ender Erdogan ◽  
Rabia Sakarya ◽  
Yasar Sakarya ◽  
Mustafa Yılmaz ◽  
...  
Keyword(s):  
Topical Administration ◽  
Corneal Neovascularization ◽  
Control Group ◽  
Therapeutic Effects ◽  
Control Groups ◽  
Group 4 ◽  
Percentage Area ◽  
Group 3 ◽  
Group 2 ◽  
Group 1

Objective. To investigate the effects of topical and subconjunctival tigecycline on the prevention of corneal neovascularization.Materials and Methods. Following chemical burn, thirty-two rats were treated daily with topical instillation of 1 mg/mL tigecycline (group 1) or subconjunctival instillation of 1 mg/mL tigecycline (group 3) for 7 days. Control rats received topical (group 2) or subconjunctival (group 4) 0.9% saline. Digital photographs of the cornea were taken on the eighth day after treatment and analyzed to determine the percentage area of the cornea covered by neovascularization. Corneal sections were analyzed histopathologically.Results. The median percentages of corneal neovascularization in groups 1 and 3 were 48% (95% confidence interval (CI), 44.2–55.8%) and 33.5% (95% CI, 26.6–39.2%), respectively. The median percentages of corneal neovascularization of groups 1 and 3 were significantly lower than that of the control group (P=0.03andP<0.001, resp.). Histologic examination of samples from groups 1 and 3 showed lower vascularity than that of control groups.Conclusion. Topical and subconjunctival administration of tigecycline seems to be showing promising therapeutic effects on the prevention of corneal neovascularization. Furthermore, subconjunctival administration of tigecycline is more potent than topical administration in the inhibition of corneal neovascularization.

A quantitative method to evaluate mesenchymal stem cell lipofection using real-time PCR

2010 ◽  
Vol 26 (5) ◽  
pp. 1501-1504 ◽  
Author(s):  
S.C. Ribeiro ◽  
R. Mendes ◽  
C. Madeira ◽  
G.A. Monteiro ◽  
C.L. da Silva ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Method

Influence of Hematopoietic Cell Lysate Vaccinations and Dendritic Cell Enriched Marrow Grafts on Engraftment after Nonmyeloablative Hematopoietic Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5231-5231
Author(s):  
Sandra Lange ◽  
Bettina Lange ◽  
Heike Vogel ◽  
Volker Weirich ◽  
Inken Hilgendorf ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Cell ◽  
Hematopoietic Chimerism ◽  
Group 3 ◽  
Group 2 ◽  
Mixed Chimeras ◽  
Group 1

Abstract Background: Stable mixed hematopoietic chimerism can be established in a canine marrow transplantation model using nonmyeloablative conditioning consisting of total body irradiation (TBI, 2Gy) and postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporine A (CSA). Reduction of TBI to 1Gy resulted in rejection of marrow grafts in this model even if CD3 depleted donor PBMC were added to the graft. Here, we investigated whether addition of immature dendritic cells or postgrafting in vivo stimulation of donor T cells against recipient derived hematopoietic antigens are capable of enhancing targeted GVH reactions and allow engraftment. Methods: Dog leukocyte antigen-identical littermates received 2Gy TBI (group 1, control, n=7) and 1Gy TBI (group 2, n=7; group 3, n=6) before allogeneic stem cell transplantation (SCT). All recipients were given postgrafting immunosuppression consisting of 15mg/kg CSA BID PO (days -1 to +35) and 10mg/kg MMF BID PO (days 0 to +27). In addition, group 2 was vaccinated ID and SQ with recipient blood cell lysates (3x/week w1-w5; 1x/week w6-w9) together with locally applied granulocyte macrophage-colony stimulating factor (125μg/vaccination). The vaccine consisted of PBMC and granulocytes at a ratio of 1:1 (105 cells/kg before lysis). Dogs of group 3 received ex-vivo generated immature monocyte-derived dendritic cells (MoDC) of donor origin in addition to marrow at day 0. MoDC doses ranged from 6.4–10.9x105 cells/kg (median 7.9x105). Littermate donor marrow was infused IV at a median of 3.4x108 (1.6–11.4x108) nucleated cells/kg. Results: The median platelet and leukocyte nadirs in group 1 were 30x109/l (median at day 11) and 2.4x109/l (median at day 8), respectively. In groups 2 and 3 the median platelet and leukocyte nadirs were 110x109/l (median at day 12) and 4.8x109/l (median at day 7). All dogs in group 1 engrafted and showed mixed hematopoietic chimerism among PBMC and granulocytes (after 4 weeks (w4): median 32% (5–44%) and 68% (10–74%), respectively). 4/7 animals remained stable mixed chimeras &gt;25 weeks, 3/7 rejected their graft (w10, w14, w16). All dogs in group 2 showed initial engraftment and chimerism of donor PBMC and granulocytes (w4: median 12% (10–19%) and 19% (11–24%), respectively), but rejected their grafts a median of 10.3 weeks (w8-w11) after SCT. 4/6 dogs in group 3 had a median w4-chimerism in PBMC and granulocytes of 14% (11–15%) and 20% (16–27%), respectively and 2/6 dogs are too early after SCT to be evaluated. Currently 3/4 evaluable dogs in group 3 remain mixed chimeric. In comparison to group 1 engraftment kinetics of groups 2 and 3 indicate a delayed engraftment combined with significantly lower donor PBMC and granulocytes chimerisms and significantly reduced engraftment duration (group 2). No differences in w4-chimerism patterns between groups 2 and 3 could be observed. Conclusion: Our data show that vaccinations with recipient hematopoietic cell lysates failed to expedite long term bone marrow engraftment following a 1Gy TBI conditioning. Addition of donor-MoDC to the graft might favour sustained engraftment but a longer follow up is needed. Furthermore, equal dosed MMF applied orally seems to be inferior to SQ applications in postgrafting immunosuppression since compared to historical controls less mixed chimeras could be generated following 2Gy TBI.

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