A rapid high-precision flow cytometry based technique for total white blood cell counting in chickens.

The automated analysis of total white blood cell count and white blood cell differentials is routine in research and clinical diagnosis in mammalian species. In contrast, in avian haematology these parameters are still estimated by conventional microscopic procedures due to technical difficulties associated with the morphological peculiarities of avian erythrocytes and thrombocytes. Both cell types are nucleated and fairly resistant to cell lysis, a prerequisite for automated leukocyte quantification and differentiation by commercial instruments. By using an anti-CD45 monoclonal antibody in combination with selected subset specific markers we have established a simple (no-lyse no-wash single-step one-tube) flow cytometry based technique for high precision chicken blood cell quantification. EDTA-blood samples are diluted, spiked with fluorescence beads and incubated with a mixture of fluorochrome conjugated chicken leukocyte specific antibodies. We demonstrate that total leukocyte numbers as well as thrombocyte, monocyte, T-cell, B-cell and heterophilic granulocyte numbers can be determined by flow cytometry in a single step without prior cell lysis, cell separation or cell washing steps. Importantly, we also show that blood samples can be fixed prior to cell staining which enables shipping of samples making the technology widely available. Comparison of this technique with conventional microscopy revealed superior precision. By comparing leukocyte differentials of two chicken populations and during immune system development after hatch we demonstrate that large sample numbers can be analysed within hours. This technique will help to overcome previous restrictions in immune status analysis in chickens in experimental systems, during vaccine testing and health status monitoring in chicken flocks. Advances in avian genomics should facilitate the development of appropriate tools for other avian species in the future which will make this technique broadly applicable.

Expression of the carbohydrate tumour marker SLeX, SLeA (CA19-9), LeY and Thomsen-Friedenreich (TF) antigen on normal squamous epithelial tissue of the penis and vagina.

BACKGROUND: Sialyl Lewis x (SLeX), sialyl Lewis a (SLeA), Lewis Y (LeY) and the Thomsen-Friedenreich (TF) antigen are carbohydrate motifs that mediate the adhesion between tumour cells and the endothelium. These antigens are usually not expressed in non-malignant tissue. Overexpression of SLeX and SLeA is combined with poor prognosis and malignant relapse. In this study, we analysed the combined expression of SLeX, SLeA, LeY and TF in normal squamous epithelium tissue of the penis shaft, glans and foreskin and in addition of the vagina and vulva. MATERIALS AND METHODS: Paraffin-embedded slides of vaginal tissue (8), vulva tissue (8) and penis shaft (8) and glans tissue (8) were fixed and incubated with monoclonal antibodies against SLeX (IgM), SLeA (IgM), LeY (IgM) and TF (IgM). Staining reaction was performed with ABC reagent. The intensity of immunohistochemical reaction on images of the slides was analyzed using a semiquantitative score. RESULTS: Strong focal expression of both sialyl Lewis antigens was found in the uretra of the penis shaft and on epithelial tissue of the glans, and permanent moderate expression of SLeX and SLeA in squamous epithelial tissue of the vagina. Moderate expression of TF was observed in male squamous epithelial tissues of the glans and foreskin and faint expression of TF was found in vulval epithelial tissue. Faint expression of Le Y was observed in female vulval epithelial tissue. CONCLUSION: Expression of SLeX, SLeA, LeY and especially of the TF antigen in normal non malignant epithelial tissue is surprising and can be explained by the function of this tissue in human reproduction. In addition, moderate TF expression seems to be restricted to epithelial tissue of the penis glans and foreskin.

Delivery of superparamagnetic nanoparticles for local chemotherapy after intraarterial infusion and magnetic drug targeting.

BACKGROUND: Superparamagnetic nanoparticles are currently used as contrast agents for magnetic resonance imaging. These particles can also be used as drug carriers for local chemotherapy, called magnetic drug targeting. Using an external magnetic field, colloidal nanoparticles can be directed to a specific body compartment (i.e. tumor). MATERIALS AND METHODS: After magnetic drug targeting in an experimental rabbit model with a VX2 squamous cell carcinoma, tumor tissue was extracted and embedded in paraffin for histology and X-ray imaging. RESULTS: The distribution of magnetic nanoparticles was detected holistically with X-ray imaging and in detail using Prussian blue staining of histological cross sections. CONCLUSION: The biodistribution of magnetic nanoparticles can be visualized with X-ray imaging and histologically confirmed.

Silica- and alkoxysilane-coated ultrasmall superparamagnetic iron oxide particles: a promising tool to label cells for magnetic resonance imaging.

In this study silica- and alkoxysilane-coated ultrasmall superparamagnetic iron oxide (USPIO) particles were synthesized, and their ability to label immortalized progenitor cells for magnetic resonance imaging (MRI) was compared. USPIO particles were synthesized by coprecipitation of ferric and ferrous salts. Subsequently, the particles were coated with silica, (3-aminopropyl)trimethoxysilane (APTMS), and [N-(2-aminoethyl)-3-aminopropyl]trimethoxysilane (AEAPTMS). The size of the USPIO particles was about 10 nm without a significant increase in diameter after coating. The highest T2 relaxivity was achieved for silica-coated USPIO particles, 339.80 +/- 0.22 s-1 mM-1, as compared with APTMS- and AEAPTMS-coated ones, reaching 134.40 +/- 0.01 and 84.79 +/- 0.02 s-1 mM-1, respectively. No toxic effects on the cells could be detected by trypan blue, TUNEL, and MTS assays. Uptake of USPIO particles was evaluated by Prussian blue staining, transmission electron microscopy, T2-MR relaxometry, and mass spectrometry. It was found that cell uptake of the different USPIO particles increased for longer incubation times and higher doses. Maximum cellular iron concentrations of 42.1 +/- 4.0 pg/cell (silica-coated USPIO particles), 37.1 +/- 3.5 pg/cell (APTMS-coated USPIO particles), and 32.7 +/- 4.0 pg/cell (AEAPTMS-coated USPIO particles) were achieved after incubation of the cells with USPIO particles at a dose of 3 micromol/mL for 6 h. The decrease of the T2 relaxation time of the cell pellets was most pronounced for cells incubated with silica-coated USPIO particles followed by APTMS- and AEAPTMS-coated particles, respectively. In gelatin gels even small clusters of labeled cells were detected by 1.5 T MRI, and significant changes in the T2 relaxation times of the gels were determined for 10000 labeled cells/mL for all particles. In summary, as compared with APTMS- and AEAPTMS-coated particles, silica-coated USPIO particles provide the highest T2 relaxivity and most effectively reduce the T2 relaxation time of immortalized progenitor cells after internalization. This suggests silica-coated USPIO particles are most suited for cell labeling approaches in MRI.

Induction of apoptosis in human breast cancer and trophoblast tumor cells by galectin-1.

Galectin-1 (gal-1), a member of the mammalian beta-galactoside-binding proteins, preferentially recognizes Galbeta1-4GlcNAc (LacNAc) sequences of oligosaccharides associated with several cell surface glycoconjugates. As demonstrated histochemically, gal-1 recognizes appropriate glycoepitopes on human breast cancer cells (MCF7) and on human chorionic carcinoma cells (BeWo). Gal-1 is expressed in many malignant and normal tissues. A high level of expression is found in lymphatic organs, which feature high rates of apoptosis. Furthermore, it is known that galectin-1 can initiate T cell apoptosis. In this study, we examined the apoptotic potential of gal-1 in vitro on MCF7 and BeWo cells. After growing both cell lines on chamber-slides for three days, apoptosis was induced by incubation with 30 microg gal-1 per ml serum-free medium for 6, 9 and 20 hours. To avoid false increased rates of apoptosis by deletion of FCS, all approaches were done with and without FCS. Apoptotic cells were detected by in situ nick translation. The rate of apoptosis was determined by counting 1500 cells per chamberslide. The normal rate of apoptosis ranged between 0.1% and 0.3%. The incubation of both cell lines with 30 microg/ml gal-1 in serum-free medium for 6 and 9 hours marginally raised the number of apoptotic cells. An increase of apoptosis was only shown by additional stimuli like hyperthermia, removal of CO2 and FCS for 20 hours. Impressing findings were manifested in an older passage of BeWo cells, in which only omission of FCS caused apoptotic rates for up to 25% after 6 hours. The presence of mycoplasma in this BeWo passage was shown by PCR. Our results demonstrated, that galectin-1 shows apoptotic potential in both the epithelial tumour cell lines examined only with additional stress stimuli.

Physical and biological characterization of superparamagnetic iron oxide- and ultrasmall superparamagnetic iron oxide-labeled cells: a comparison.

RATIONALE: Superparamagnetic iron-oxide particles are used frequently for cellular magnetic resonance imaging and in vivo cell tracking. The purpose of this study was to compare the labeling characteristics and efficiency as well as toxicity of superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) for 3 cell lines. METHODS: Using human fibroblasts, immortalized rat progenitor cells and HEP-G2-hepatoma cells, dose- and time-dependence of SPIO and USPIO uptake were evaluated. The amount of intracellular (U)SPIO was monitored over 2 weeks after incubation by T2-magnetic resonance relaxometry, ICP-mass-spectrometry, and histology. Transmission-electronmicroscopy was used to specify the intracellular localization of the endocytosed iron particles. Cell death-rate and proliferation-index were assessed as indicators of cell-toxicity. RESULT: For all cell lines, SPIO showed better uptake than USPIO, which was highest in HEP-G2 cells (110 +/- 2 pg Fe/cell). Cellular iron concentrations in progenitor cells and fibroblasts were 13 +/- 1pg Fe/cell and 7.2 +/- 0.3pg Fe/cell, respectively. For all cell lines T2-relaxation times in cell pellets were below detection threshold (<3 milliseconds) after 5 hours of incubation with SPIO (3.0 micromol Fe/mL growth medium) and continued to be near the detection for the next 6 days. For both particle types and all cell lines cellular iron oxide contents decreased after recultivation and surprisingly were found lower than in unlabeled control cells after 15 days. Viability and proliferation of (U)SPIO-labeled and unlabeled cells were not significantly different. CONCLUSIONS: The hematopoetic progenitor, mesenchymal fibroblast and epithelial HEP-G2 cell lines accumulated SPIO more efficiently than USPIO indicating SPIO to be better suited for cell labeling. However, the results indicate that there may be an induction of forced cellular iron elimination after incubation with (U)SPIO.

Triple-coated stents (Hirudin/Iloprost/Paclitaxel): an in vitro approach for characterizing the antiproliferative potential of each individual compound.

BACKGROUND: Hirudin (H)/iloprost (I)/paclitaxel (P)-coated stents represent a multifactorial approach to reducing the proliferative response caused by ballooning and stenting. The study presented compares the net effect of each individual compound of HIP-coated stents with the summed effect of the compounds in the stent coating. METHODS AND RESULTS: For proliferation prescreening studies, human coronary smooth muscle cells were incubated with H (0.005-500 microg/ml), I (0.00001-1 microg/ml), and P (0.0001-10 microg/ml). After 5 days, cell number was studied in a cell analyzer system. Secondly, 8-mm stents were coated with (1) HI, (2) HIP-10 microg/20 microg/40 microg (HIP5%/10%/20%), (3) P-40 microg (P), (4) IP-40 microg (IP), and (5) HP-40 microg (HP). After 5 days, the effect on cell proliferation and cytoskeletal structures was studied. No antiproliferative effect was found after incubation with H; significant inhibition was seen after incubation with I (p<0.05) or lipophilically dissolved P (p<0.001). After 5 days incubation with HIP5%-, HIP10%-, HIP20%-, P20%-, IP20%-, and HP20%-coated stents, cell proliferation was inhibited by 55.5% (p<0.05), 61% (p<0.05), 57.9% (p<0.05), 59.5% (p<0.001), 59.8% (p<0.001), and 63.3% (p<0.001), respectively. HI- and HIP-coated stents caused a severe destruction of the cytoskeletal structures smooth muscle alpha-actin and alpha-tubulin; despite the destruction, vital cells could be identified with positive FDA staining. CONCLUSIONS: Although both lipophilically dissolved P and hydrophilically dissolved I contributed to the antiproliferative effect, no additive effect of the two compounds was detected. In vivo P can be released more easily from the coating material due to the permanent lipophilic contact of the stent struts with the vessel wall. The current study is the first report on a clear and uncomplicated technique to obtain information on the antiproliferative potential of coated stents before large experimental studies are initiated.

Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4.

The homing and tissue-specific recruitment of bone marrow-derived progenitor cells is a major issue in stem cell research and therapy. Chemokine biology plays a central role in the homing and trafficking of leukocytes. Here we show functional expression of the chemokine receptors CCR1, CCR4, CCR7, CCR10, and CXCR5 on primary isolates of CD34- mesenchymal progenitor cells as well as immortalized mesenchymal stem cell (MSC) lines. Although mRNA expression of CXCR4 was detected in both primary cells and immortalized clones, the receptor was not expressed on the cell surface. On the basis of this expression profile, the MSC could potentially home to secondary lymphatic organs (CCR7, CXCR5), skin (CCR4, CCR10), small intestine (CCR10), and salivary glands (CCR10). To study tissue-specific homing, murine CD34- MSC lines showing concordant chemokine receptor expression were either transiently labeled with CMFDA, or were stably transfected with green fluorescent protein (GFP) expression plasmids. The MSC were then injected into syngeneic healthy mice, and the distribution of the cells determined. The injected cells efficiently homed to spleen, thymus, and lymph nodes. In addition, cells were found in the mucosa of the small intestine, skin, and salivary gland. No significant recruitment to bone marrow, liver, or kidney was seen. Chemokine biology may play an important role in the homeostasis and potentially tissue recruitment of early adult progenitor cells.

Improved arteriogenesis with simultaneous skeletal muscle repair in ischemic tissue by SCL(+) multipotent adult progenitor cell clones from peripheral blood.

BACKGROUND: The CD34(-) murine stem cell line RM26 cloned from peripheral blood mononuclear cells has been shown to generate hematopoietic progeny in lethally irradiated animals. The peripheral blood-derived cell clones expresses a variety of mesodermal and erythroid/myeloid transcription factors suggesting a multipotent differentiation potential like the bone marrow-derived 'multipotent adult progenitor cells' (MAP-C). METHODS: SCL(+) CD34(-) RM26 cells were transfused intravenously into mice suffering from chronic hind-limb ischemia, evaluating the effect of stem cells on collateral artery growth and simultaneous skeletal muscle repair. RESULTS: RM26 cells are capable of differentiating in vitro into endothelial cells when cultured on the appropriate collagen matrix. Activation of the SCL stem cell enhancer (SCL(+)) is mediated through the binding to two Ets and one GATA site and cells start to express milieu- and growth condition-dependent levels of the endothelial markers CD31 (PECAM) and Flt-1 (VEGF-R1). Intravenously infused RM26 cells significantly improved the collateral blood flow (arteriogenesis) and neo-angiogenesis formation in a murine hind-limb ischemia transplant model. Although transplanted RM26 cells did not integrate into the growing collateral arteries, cells were found adjacent to local arteriogenesis, but instead integrated into the ischemic skeletal muscle exclusively in the affected limb for simultaneous tissue repair. CONCLUSION: These data suggest that molecularly primed hem-/mesangioblast-type adult progenitor cells can circulate in the peripheral blood improving perfusion of tissues with chronic ischemia and extending beyond the vascular compartment.