Effect of various platelet preparations on retinal muller cells.

PURPOSE: Peeling of the internal limiting membrane is the treatment of choice for macular holes. Fresh platelet suspension (PS) is used to support wound healing in persistent macular holes. The concentration of growth factors in fresh, frozen, and thrombin-activated PSs were compared, to optimize their trophic potential and examine their capacity to support proliferation, migration, and contraction of human retinal Müller cells. METHODS: The concentration of various growth factors in frozen PS, thrombin-activated PS, and plasma were evaluated by ELISA. The effect of these preparations on proliferation, migration, and contraction of human Müller cells were evaluated with an ATP-assay, a colony-dispersion assay, and a detached collagen gel contraction assay respectively. Plasma was tested as a control. RESULTS: Frozen and thrombin-activated PSs contained significantly more EGF, TGF-beta1, and PDGF than did plasma. The highest concentrations of EGF and FGF were found in frozen PS. All platelet preparations and plasma supported cell growth significantly better than the control, which was serum-free culture medium. Müller cells migrated better when incubated with thrombin-activated PS than with any other test solution. Contraction was extremely strong after incubation with fresh PS compared with plasma or thrombin-activated or frozen PSs. CONCLUSIONS: Frozen and thrombin-activated PSs may be suitable alternatives to fresh PS for persisting macular holes, due to their superior effect on Müller cell migration.

In vitro proliferation and differentiation of human CD34+ cells from peripheral blood into mature red blood cells with two different cell culture systems.

BACKGROUND: An in vitro erythropoiesis assay is a powerful tool for investigating red blood cell (RBC) development and diseases of the erythroid lineage. Most assays, however, failed in either proliferation or terminal differentiation. Here two liquid cultures (LCs) for in vitro generation of RBCs from peripheral blood CD34+ cells were compared. STUDY DESIGN AND METHODS: Granulocyte-colony-stimulating factor-mobilized CD34+ cells were cultured for 16 days in a two-phase LC (2P-LC; Days 1-8, stem cell factor [SCF], erythropoietin [EPO], insulinlike growth factor [IGF]-1, and steroids; Days 9-16, EPO and insulin) and for 21 days in a three-phase LC (3P-LC; Days 1-7, SCF, thrombopoetin, and Flt3-ligand; Days 8-14, SCF, EPO, and IGF-1; Days 15-21, EPO and IGF-1). Maturation was analyzed by flow cytometry (CD36, CD71, glycophorin A [GPA]) and microscopy. RESULTS: In the 2P-LC, cell numbers increased from 0.5 x 10(6) to 25.7 x 10(6) +/- 15.1 x 10(6) cells per mL. More than 95 percent were GPA+ and showed morphologic characteristics of normoblasts (52 +/- 15%) and enucleated reticulocytes (43 +/- 18%). In the 3P-LC, a higher overall proliferation to 55.7 x 10(6) +/- 37.7 x 10(6) cells per mL was achieved (p < 0.05). This was also accompanied by a high degree of normoblasts (36 +/- 16%) and reticulocytes (48 +/- 24%). The amount of GPA+ cells was slightly lower (88.4 +/- 16.4%), associated with a significantly higher contamination by nonerythroid cells (15.8 +/- 19.3% vs. 3.9 +/- 2.9%, p < 0.05). CONCLUSION: Both LCs were able to generate fully matured RBCs and represent powerful tools for fundamental research in erythroid development and diseases targeting the erythroid lineage. A slightly higher proliferation was achieved in the 3P-LC. This was associated with a limited homogeneity due to more nonerythroid cells, however. Therefore the 2P-LC is favored, also saving additional culture days and growth factors.

Corneal epitheliotrophic capacity of three different blood-derived preparations.

PURPOSE: Serum eye drops have been successfully used in the treatment of severe ocular surface disorders. Fresh frozen plasma (FFP) and platelet concentrates have not yet been tested for use as eye drops, although they are easily available as quality-controlled products from blood banks and are routinely used for transfusion. To test whether FFP or platelet-derived growth factor solutions could be used for ocular surface diseases, we compared the epitheliotrophic capacity of platelet releasate and FFP with that of serum in cell culture models. METHODS: The concentrations of EGF, TGF-beta1, PDGF-AB, fibronectin, vitamin A and vitamin E in serum, FFP, and platelet releasate were evaluated with ELISA and HPLC. Corneal epithelial cells were incubated with the various preparations and cell proliferation, migration, and differentiation were evaluated by means of a luminescence-based adenosine triphosphate (ATP) assay, a colony dispersion assay, and scanning electron microscopy. RESULTS: Growth factor concentrations were significantly higher in platelet releasate than in serum and were lowest in FFP. Fibronectin and vitamins were found in higher concentrations in serum than in FFP and were lowest in platelet releasate. Cell proliferation was best supported by platelet releasate followed by serum and FFP; however, cell migration and differentiation were better supported by serum than by platelet releasate and FFP. The reduced nutrient capacity of FFP was in part found to be due to an antiproliferative effect of citrate used as an anticoagulant in the production process. CONCLUSIONS: Platelet releasate but not FFP may offer additional potential for the treatment of severe ocular surface disease. Platelet releasate may be suitable as a novel treatment option for ocular surface disease with a superior effect on cell growth.

Epitheliotrophic capacity of a growth factor preparation produced from platelet concentrates on corneal epithelial cells: a potential agent for the treatment of ocular surface defects?

BACKGROUND: Topical application of serum eye drops has been reported to accelerate healing of persistent ocular surface defects. It is supposed that growth factors in serum support the wound healing process. Platelets (PLTs) are rich in growth factors and easily available as PLT concentrates (PCs) from blood banks. Therefore, growth factor preparations from PCs may serve as a new and superior therapeutic agent for such defects. STUDY DESIGN AND METHODS: After thrombin stimulation for growth factor release, the cell-free supernatant (PLT releasate) of washed PCs (n = 8) was analyzed for epitheliotrophic factors and its wound healing capacity in comparison to serum (n = 8). Human corneal epithelial cells were used as a model to investigate cell growth, migration, and differentiation in response to both blood products. RESULTS: PLT releasate contains more epithelial growth factor, PLT-derived growth factor, and transforming growth factor-beta, but less hepatocyte growth factor, fibronectin, and vitamins. Cell growth was significantly better in response to PLT releasate. Migration and differentiation were slightly better supported by serum. CONCLUSION: Possibly owing to its high content of growth factors, PLT releasate has a distinct superior effect on cell growth. Stimulation of migration and differentiation was slightly inferior but still acceptable. PLT releasate could therefore be a novel treatment option for ocular surface defects.

Interferon-alpha stimulation of liver cells enhances hepatitis delta virus RNA editing in early infection.

BACKGROUND/AIMS: RNA editing controls the formation of hepatitis-delta-antigen-S and -L and therefore plays a central role in the hepatitis-delta-virus (HDV) life-cycle. Editing is catalyzed by the enzyme Adenosine-deaminase-acting-on-RNA1 (ADAR1) of which two different forms, ADAR1-L and ADAR1-S, exist. As ADAR1-L is induced by interferon (IFN)-alpha, we examined the influence of IFN-alpha-stimulation of host cells on HDV-RNA editing. METHODS: Editing was studied in Huh-7-cells transfected with HDV-RNA on days 7, 14, 21 and 28 after transfection. ADAR1-L mRNA was measured by RT-PCR. RESULTS: IFN-alpha-treatment led to a 5-fold higher expression of ADAR1-L and to an increase in editing from 14+/-2% (SD) in unstimulated controls to 27+/-4% (SD) on day 7 after transfection. Editing further increases over time to the same maximum level of 35% in IFN-alpha-treated as well as untreated cells. CONCLUSIONS: By IFN-alpha-stimulation both ADAR1-L expression and editing are increased in Huh-7-cells at day 7, and the maximum level of edited antigenomes is reached earlier with IFN-alpha-treatment as compared to untreated cells. Thus, ADAR1-L appears to be able to increase editing, but the HDV genome apparently has an intrinsic negative feed-back regulation mechanism that limits editing to roughly a third of the genomes.

Recommendations for optimized settings of the Amicus Crescendo cell separator for the collection of CD34+ progenitor cells.

BACKGROUND: CD34+ PBPCs for autologous transplantation purposes are collected by leukapheresis procedures on automated cell separators. In this study, the influence of different parameters on collection efficiency (CE) of the Amicus Crescendo cell separator (Baxter) was investigated. STUDY DESIGN AND METHODS: A total of 146 PBPC collections with Amicus cell separators were performed in 56 patients with either settings recommended by the manufacturer or modified settings to identify variables that have a significant and important impact on CE. RESULTS: By use of a standard setting with a cycle volume of 1400 mL, CE significantly decreases when patients' preapheresis peripheral blood WBC counts are between 25,000 and 35,000 per micro L. CE can be improved if cycle volume is reduced to 1000 mL. If WBC concentrations exceed 55,000 per micro L before apheresis, CE also significantly decreases despite of reduced cycle volume. Additionally, high flow rates greater than 60 mL per minute significantly reduce CE. CONCLUSION: Parameters influencing the outcome of CD34+ PBPC collections were identified, such as patients' WBC count, cycle volume, and whole blood flow rate. An optimized adjustment of these variables will further increase the CE of the device.

Diseased vein grafts express elevated inflammatory cytokine levels compared with atherosclerotic coronary arteries.

BACKGROUND: The pathologic modifications characterizing vein graft disease resemble those observed in native arteriosclerosis, but in accelerated form. Although both disorders are considered to be inflammatory diseases, it remains to be determined whether diseased vein grafts and atherosclerotic coronary arteries differentially express inflammatory mediators. Therefore, we examined whether differences in the expression of proinflammatory cytokines by these two distinct vascular pathologies favor the accelerated inflammation within diseased vein grafts. METHODS: The messengerRNA expression of various cytokines (interleukin-1 beta [IL-1 beta], IL-6, IL-8, tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma]) was quantified using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in tissue samples of native saphenous veins (NSV, n = 5), diseased coronary arteries (CAD, n = 25), and diseased vein grafts (VG, n = 13). RESULTS: Native saphenous veins did not contain any detectable transcripts except for IFN-gamma. As expected, CAD was characterized by the expression of IL-1 beta, IL-6, IL-8, IFN-gamma, and TNF-alpha mRNA. Interestingly VG also expressed these mediators, but at markedly higher levels. Quantification by RT-PCR revealed that, compared with specimens from the CAD group, VG specimens contained 5.8 +/- 1.2 times, 286 +/- 22 times, and 29 +/- 7.3 times as many transcripts for the cytokines IL-1 beta, IL-6 and TNF-alpha, respectively, as well as 25 +/- 8.3 times more transcripts for the chemokine IL-8. In contrast, the expression of IFN-gamma transcripts did not differ among the groups. CONCLUSIONS: The elevated expression of proinflammatory cytokine transcripts supports the hypothesis that diseased vein grafts, compared with atherosclerotic coronary arteries, are characterized by enhanced inflammatory activity that might accelerate atherosclerotic modifications. This may implicate new therapeutic strategies for the prevention of vein graft disease.