A comparative study of PKH67, DiI,and BrdU labeling techniques for tracing rat mesenchymal stem cells

Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 μM BrdU and 10 μM BrdU. The cells were then cultured for 6 d or passaged (1–3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3?±?1.6% PKH67+ and 98.4?±?1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2?±?4.6% 10 μM BrdU+ and 77.6?±?4.6% 5 μM BrdU+), which remained high for 6 d. Viability of labeled cells was 91?±?3.8% PKH67+, 90?±?1.5% DiI+, 91?±?0.8% 5 μM BrdU+, and 76.9?±?0.9% 10 μM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.     

Lack of Tryptophan Hydroxylase-1 in Mice Results in Gait Abnormalities

The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (−/−) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system.     

Comparison of Manual and Automatic (MagNA Pure) Isolation Methods of Total RNA from Adipose Tissue

Aim Comparison of manual and automatic (MagNA Pure) isolation methods of total RNA from adipose tissue with respect to its quality and recovery factor. Material 120 human subcutaneous adipose tissue samples (about 100 mg/sample) were collected from patients during surgical operations. The tissue sample was stabilized in RNAlater (QIAGEN GmbH, Germany). Methods Total RNA was extracted by the following kits: Rneasy Protect Mini, Rneasy Lipid Tissue (QIAGEN GmbH, Germany) and MagNA Pure Compact RNA Isolation (Tissue) for MagNA Pure Compact Instrument (Roche Diagnostics GmbH, Germany). Results The average RNA yields with Rneasy Lipid Tissue kits were about two-fold higher in comparison with the Rneasy Protect Mini kit. When the MagNA Pure Compact System was used, RNA yields from the same sample were more uniform compared with manual systems. It was also more convenient and less time-consuming than the manual approach. No DNA contamination of total RNA samples was detected except for samples isolated by Rneasy Protect Mini Kit. Conclusion Rneasy Lipid Tissue Kit and MagNA Pure Compact RNA Isolation Kit (Tissue) provide RNA samples of high quantity, purity and PCR amplificability. RNA samples are suitable for further processing using methods of molecular biology.     

Fluconazole and itraconazole susceptibility of vaginal yeast isolates from Slovakia

Vulvovaginal candidiasis is a common mucosal infection caused by opportunistic yeasts of the Candida genus. In this study, we isolated and identified the yeast species in the vagina of patients treated in the gynecology clinic and tested in vitro activities of fluconazole and itraconazole against 227 clinical yeast isolates by the NCCLS microdilution method. C. albicans (87.6%) was the most frequently identified species followed by C. glabrata (6.2%) and C. krusei (2.2%). Almost thirteen percent of yeast strains were resistant to fluconazole and 18.5% were resistant to itraconazole. Cross-resistance analyses of C. albicans isolates revealed that fluconazole resistance and itraconazole resistance were also associated with decreased susceptibilities to other azole derivatives mainly to ketoconazole and miconazole. At the same time no cross-resistance to polyene antibiotics amphotericin B and nystatin was observed. These results support the notion that antifungal agents used to treat vaginitis may be contributing to the drug resistance problem by promoting cross-resistance to a range of clinically used antifungals.     

A direct role for C1 inhibitor in regulation of leukocyte adhesion

Plasma C1 inhibitor (C1INH) is a natural inhibitor of complement and contact system proteases. Heterozygosity for C1INH deficiency results in hereditary angioedema, which is mediated by bradykinin. Treatment with plasma C1INH is effective not only in patients with hereditary angioedema, but also in a variety of other disease models, in which such therapy is accompanied by diminished neutrophil infiltration. The underlying mechanism has been explained primarily as a result of the inhibition of the complement and contact systems. We have shown that C1INH expresses the sialyl-Lewis(x) tetrasaccharide on its N-linked glycan, via which it binds to E- and P-selectins and interferes with leukocyte-endothelial adhesion in vitro. Here we show that both native C1INH and reactive center cleaved C1INH significantly inhibit selectin-mediated leukocyte adhesion in several in vitro and in vivo models, whereas N-deglycosylated C1INH loses such activities. The data support the hypothesis that C1INH plays a direct role in leukocyte-endothelial cell adhesion, that the activity is mediated by carbohydrate, and that it is independent of protease inhibitory activity. Direct involvement of C1INH in modulation of selectin-mediated cell adhesion may be an important mechanism in the physiologic suppression of inflammation, and may partially explain its utility in therapy of inflammatory diseases.     

Comparative In Vivo Effects of Hemoglobin-Based Oxygen Carriers (HBOC) with Varying Prooxidant and Physiological Reactivity

A series of hemoglobin-based oxygen carrier candidates (HBOC), previously noted for their differences in prooxidative and physiological reactivity, were compared in terms of the negative effects displayed upon injection in Wistar rats. At the concentrations tested, antioxidant strategies based on albumin as well as based on rubrerythrin appear to offer observable physiological advantages.     

CD40L stabilizes arterial thrombi by a beta3 integrin--dependent mechanism

CD40L, a member of the tumor necrosis factor family of ligands, plays a major role in immune responses via its receptor, CD40. Recently, CD40L has been detected on the surfaces of activated platelets and shown to activate endothelium. Here we further addressed the function of platelet CD40L. We show that absence of CD40L affects the stability of arterial thrombi and delays arterial occlusion in vivo. Infusion of recombinant soluble (rs)CD40L restored normal thrombosis, whereas rsCD40L lacking the KGD integrin-recognition sequence did not. CD40-deficient mice exhibited normal thrombogenesis. rsCD40L specifically bound to purified integrin alphaIIbbeta3 and to activated platelets in a beta3-dependent manner and induced platelet spreading. In addition, rsCD40L promoted the aggregation of either human or mouse platelets under high shear rates. Thus, CD40L appears to be an alphaIIbbeta3 ligand, a platelet agonist, and necessary for stability of arterial thrombi.     

Iron-independent specific protein expression pattern in the liver of HFE-deficient mice

Hereditary hemochromatosis type I is an autosomal-recessive iron overload disease associated with a mutation in HFE gene. The most common mutation, C282Y, disrupts the disulfide bond necessary for the association of HFE with beta-2-microglobulin and abrogates cell surface HFE expression. HFE-deficient mice develop iron overload indicating a central role of the protein in the pathogenesis of hereditary hemochromatosis type I. However, despite significant effort, the role of the HFE protein in iron metabolism is still unknown. To shed a light on the molecular mechanism of HFE-related hemochromatosis we studied protein expression changes elicited by HFE-deficiency in the liver which is the organ critical for the regulation of iron metabolism. We undertook a proteomic study comparing protein expression in the liver of HFE deficient mice with control animals. We compared HFE-deficient animals with control animals with identical iron levels obtained by dietary treatment to identify changes specific to HFE deficiency rather than iron loading. We found 11 proteins that were differentially expressed in the HFE-deficient liver using two-dimensional electrophoresis and mass spectrometry identification. Of particular interest were urinary proteins 1, 2 and 6, glutathione-S-transferase P1, selenium binding protein 2, sarcosine dehydrogenase and thioredoxin-like protein 2. Our data suggest possible involvement of lipocalins, TNF-alpha signaling and PPAR alpha regulatory pathway in the pathogenesis of hereditary hemochromatosis and suggest future targeted research addressing the roles of the identified candidate genes in the molecular mechanism of hereditary hemochromatosis.