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PubMed2006

[Photodynamic effect of hematoporphyrin monomethyl ether on ovarian cancer cell line SKOV3].

BACKGROUND & OBJECTIVE: Photodynamic therapy (PDT) is a new treatment choice for ovarian carcinoma. Hematoporphyrin monomethyl ether (HMME) is a novel photosensitive reagent developed in China. This study was to investigate the photodynamic effect of HMME-based PDT on human epithelial ovarian cancer cell line SKOV3. METHODS: After an incubation with 30 microg/ml HMME for different time, the fluorescent image and intracellular location of HMME in SKOV3 cells were observed under a fluorescent microscope and laser scanning confocal microscope (LSCM). After being treated with different doses (5-50 microg/ml) of HMME and irradiated with different optical doses (1.5-12 J/cm(2)) of laser, the survival rate of SKOV3 cells was measured by MTT assay. Mechanisms of cell death during PDT was determined by Annexin V/PI double staining technique and analyzed by flow cytometry. RESULTS: Red fluorescence appeared shortly after administration of HMME and localized in cytoplasm; intracellular fluorescence intensity reached the peak after 3 h. High concentrations of HMME alone had cytotoxicity to SKOV3 cells, while laser irradiation alone had no effect on cell survival. Survival rate of SKOV3 cells was gradually decreased along with the increase of HMME concentration and laser dose, but such a trend diminished when HMME concentration reached 40 microg/ml. After treatment of HMME, the dead cells were predominantly necrosis cells. CONCLUSION: HMME has a photodynamic effect on SKOV3 cells.

Cell Line, Tumor↗
PubMed2006

Mitochondrial localization and activity of P-glycoprotein in doxorubicin-resistant K562 cells.

It is now well-established that P-glycoprotein 170 (P-gp), an efflux pump involved in multidrug resistance (MDR) is overexpressed at the plasma membrane of doxorubicin-resistant K562 leukemia cells. Nevertheless, several results suggested: (i) that P-gp-mediated drug efflux was not the only mechanism involved in resistance; (ii) that intracellular compartments could accumulate the drug, preventing it from reaching its nuclear targets; (iii) that agents able to reverse multidrug resistance may lead to intracellular drug redistribution. We have studied the localization of P-gp in mitochondria as well as its functional properties in this compartment. Using several monoclonal antibodies (MoAbs) directed against different P-gp epitopes, a protein was detected in the cytoplasm of two doxorubicin-resistant K562 sublines and, by confocal laser scanning microscopy, this protein was shown to co-localize in the Golgi apparatus and in mitochondria, in equivalent proportions. Purified mitochondria were isolated from K562 cell variants; the presence of a protein of about 170 kDa and reacting with several anti-P-gp antibodies was assessed in MDR cells by Western blotting and flow cytometry. Functional assays have shown that mitochondrial P-gp was involved in doxorubicin accumulation inside the organelle but not in its efflux, suggesting an orientation of P-gp in the mitochondrial membrane inverse to that observed in the plasma membrane. A potential role for mitochondrial P-gp in MDR cells would be to protect the nucleus from doxorubicin. This is the first demonstration of the presence and functional activity of P-gp in mitochondria of MDR cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Preparation and characterization of biodegradable nanoparticles entrapping immunodominant peptide conjugated with PEG for oral tolerance induction.

PEG-conjugated immunodominant peptides for collagen-induced arthritis (CIA) were prepared for oral tolerance induction instead of whole Type II collagen (CII), because a small peptide can be converted to a macromolecule soluble in methylene chloride by the coupling of poly-ethylene glycol (PEG). PEG-pep1 was synthesized from a peptide and mPEG-NH2 (Mw approximately 5000) using SPDP as a linker, whereas PEG-pep2 was prepared by the direct disulfide coupling between PEG-OD (Mw approximately 10,000) and the peptide. PEG-pep1 and PEG-pep2 were purified by gel permeation chromatography (GPC), and the peak fractions of GPC were identified by GPC and MALDI-TOF mass spectroscopy. The peptide coupling gave much earlier retention times for PEG-pep1 (11.26 min) and PEG-pep2 (10.61 min) than for mPEG-SPDP (15.63 min) and mPEG-OD (14.58 min). The Mw's of mPEG-NH2, mPEG-SPDP, PEG-pep1, mPEG-OD and PEG-pep2 were 5451, 5588, 7035, 10,360 and 11,826, respectively, suggesting that PEG-pep1 and PEG-pep2 of high purity could be obtained. The nanoparticles entrapping PEG-pep1 and PEG-pep2 (NP/PEG-pep1 and NP/PEG-pep2) were prepared by the o/w solvent evaporation method, whereas the peptide-loaded nanoparticles (NP/pep) were prepared by the w/o/w double emulsion method. Although all the nanoparticles had a similar spherical morphology under scanning electron microscopy, NP/pep showed up as having a larger mean size than the others, which was confirmed by dynamic light scattering analysis (NP/pep, 499.7+/-27.2 nm; NP/PEG-pep1, 333.0+/-16.8 nm; NP/PEG-pep2, 342.4+/-15.1 nm). The lower encapsulation efficiency of NP/pep (21.0+/-1.6%) than NP/PEG-pep1 (66.5+/-5.0%) and NP/PEG-pep2 (73.8+/-5.5%) can also be attributed to the preparation method. In in vitro release studies, NP/PEG-pep1 and NP/PEG-pep2 displayed a similar release profile, close to a linear release pattern, whereas NP/pep displayed a tri-phasic release profile. From these results, it was demonstrated that nanoparticles entrapping a PEG-conjugated peptide could be an alternative delivery method for the induction of oral tolerance rather than CII and peptide.

Animals↗
PubMed1997

Enumeration of CD34+ hematopoietic progenitor cells in peripheral blood and leukapheresis products by microvolume fluorimetry: a comparison with flow cytometry.

There is increasing interest in both standardization and simplification of methods for enumeration of CD34+ hematopoietic progenitor cells (HPC) to facilitate cellular therapies and to improve interinstitutional comparison of clinical and laboratory results. We evaluated a novel method for CD34+ cell enumeration based on microvolume fluorimetry (MVF) compared with our laboratory's routine flow cytometric method on samples of peripheral blood and leukapheresis products. The MVF method is semiautomated and uses a 633-nm light from a helium-neon laser to scan fluorochrome-labeled cells held in stasis in a capillary known volume. The performance of the MVF assay for enumeration of CD34+ cells was found to be comparable to our routine flow cytometric assay in linearity and accuracy in the range of 5-1500 cells per microliter. Precision of MVF for replicate assays on the same instrument was demonstrated by coefficient of variation (CV) values of 8.4% at a CD34+ cell concentration of 284/microliters for a sample volume of 0.8 microliters, and 15.7% at 12/microliters for a sample volume of 3.2 microliter. Precision among three different instruments was demonstrated, using sample volumes of 1.6 microliters, by CV values of 44% at 6 cells/microliters and 4.6% at 733 cells/microliters. In a field sample evaluation, precision of the entire assay system for paired measurements on 0.8-microliter sample volumes was demonstrated by CV values of 50%, 31%, and 15% for peripheral blood samples with concentrations of 0-10, 10-20, and 20-100 CD34+ cells/microliters, respectively, and 6.3%, 8.1% and 6.5% for leukapheresis samples with concentrations of 0-100, 100-1,000, and 1,000-2,500 CD34+ cells/microliters, respectively. The MVF assay was easy to perform, required minimal technical training time, and had a turnaround time of 40 min, of which less than 10 min was actual technical time. These observations suggest that the MVF method for CD34+ cell enumeration may prove useful to clinical laboratories providing support for HPC collection, processing, and transplantation services that require relatively simple, rapid assays for product quality control or to guide real-time clinical decisions.

Antigens, CD34↗
PubMed2000

Nucleoside diphosphate kinase beta (Nm23-R1/NDPKbeta) is associated with intermediate filaments and becomes upregulated upon cAMP-induced differentiation of rat C6 glioma.

Nucleoside diphosphate kinases (Nm23/NDPK) are enzymes functional in cell proliferation, differentiation, development, tumor progression, and metastasis. Nevertheless, no consensus exists about the molecular mechanism by which Nm23/NDPK isoforms exert their role in these processes. We investigated the expression of the rat Nm23-R1/NDPKbeta and Nm23-R2/NDPKalpha isoforms, homologues of the human Nm23-H1/NDPK A and Nm23-H2/NDPK B proteins, respectively, upon cAMP-induced differentiation of rat C6 glioma cells and demonstrated a differential interaction with intermediate filaments. Semiquantitative RT-PCR, immunoblotting, and flow cytometry showed a constitutive expression of both Nm23 isoforms. After induction of differentiation in C6 cells with cAMP analogs or isoproterenol, a dose-dependent 2- and 2.5-fold upregulation of the Nm23-R1 mRNA and protein, respectively, was observed. In contrast, the expression of Nm23-R2 remained unchanged. Localization of both isoforms with confocal laser scanning microscopy demonstrated a punctate reticular staining pattern for both Nm23 isoforms in the cytosol and processes of the cells which was particularly intense in the perinuclear region. In addition, while Nm23-R2 was colocalized and coimmunoprecipitated with vimentin in nondifferentiated cells, both isoforms were associated with GFAP in differentiated cells. The significance of these findings in relation to a possible function of Nm23 isoforms in cell proliferation, differentiation, and tumor-associated mechanisms is discussed.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMedDate not supplied

In situ collagen gelation: a new method for constructing large tissue in rotary culture vessels.

In situ collagen gelation is a method that combines a static three-dimensional culture technique with rotating bioreactors. This method was designed for large dense tissue engineering ex vivo. To challenge the current limitations on size, we combined the static collagen gel embedding method with high-aspect ratio rotating bioreactors. Rat calvarial cells in gelated collagens were cultured in rotating vessels with 5 mM beta-glycerophosphate-containing medium for 1, 2, or 3 wk and then analyzed for cell morphology, cell distribution, and viability, as well as for contraction of the collagen gel. The size of collagen gels with rat calvarial cells averaged 2.8 cm in diameter x 0.25 cm in thickness at the end of 3 wk. Scanning electron microscopy and laser scanning confocal microscopy of collagen gels revealed a homogeneous distribution of living cells. Despite the barrier effects from induced calcification, in collagen gels, cell metabolic activity (alkaline phosphatase assay and 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide assay) increased over the 3 wk, and cell viability (trypan blue exclusion and flow cytometry analysis) remained at about 90% at the end of 3 wk. Based on our results, we determined that in situ collagen gelation provides a feasible method for engineering large dense tissue ex vivo.

Animals↗
PubMed2005

Effects of transforming growth factor beta 1 on the growth of rhabdomyosarcoma cell line RD.

BACKGROUND: Transforming growth factor-beta (TGF-beta) can inhibit the growth of most epithelial and endothelial cells. The growth regulative role of TGF-beta on soft tissue sarcoma was seldom reported. Here we examined TGF-beta1 effects on the growth of human rhabdomyosarcoma cell RD and searched the relative molecular mechanism. METHODS: The viability of RD was examined by [(3)H]-thymidine incorporation and [3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay. RD cell cycle was analysed by flow cytometry. The protein and mRNA of cell cycle regulative factors in RD were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The kinase activity of cdk2 or cdk4 was examined by immunoprecipitation and kinase assay. Immunofluorescent staining was used to detect the location of cell cycle regulative factors in RD by laser scanning confocal microscope. RESULTS: TGF-beta1 inhibits RD proliferation by G1-arrest in cell cycle progression. TGF-beta1 can prominently up-regulate P27 of RD, then augment P27 to bind cyclinE-cdk2 complexes, which effectively suppress cdk2 kinase activity. P21 increased and c-myc decreased in RD due to TGF-beta1. Both P15 and cdk4 have not been involved in the growth inhibitory event. TGF-beta1 treatment induced P27 to congregate around nucleus. P21 pervaded from nucleus to both nucleus and cytoplasm by TGF-beta1 treatment. CONCLUSION: TGF-beta1 inhibits the proliferation of human rhabdomyosarcoma cell line RD and induces RD G1-arrest. This course is accomplished by TGF-beta1 up-regulating P27 to suppress cdk2 kinase activity. The induction of P21 and down-regulation of C-myc might participate in the growth-arrest event.

Cell Cycle Proteins↗
PubMed2006

Quality assessment of confocal microscopy slide-based systems: instability.

BACKGROUND: All slide-based fluorescence cytometry detections systems basically include an excitation light source, intermediate optics, and a detection device (CCD or PMT). Occasionally, this equipment becomes unstable, generating unreliable and inferior data. METHODS: A number of tests have been devised to evaluate equipment performance and instability. The following four instability tests are described: galvanometer scanning, stage drift, correct wavelength spectral detection, and long-term laser power. RESULTS: Quality assurance tests revealed that a confocal microscope can become unstable in the following parameters, yielding inaccurate data: laser power, PMTs functionality, spectrophotometer accuracy, galvanometer scanning and laser stability, and stage drift. Long-term laser power stability has been observed to vary greatly. CONCLUSIONS: Confocal systems can become unstable in the following parameters: long-term laser power, galvanometer scanning, spectrophotometer accuracy, and stage stability. Instability in any of these parameters will affect image quality. Laser power fluctuations result from either a defective Acousto-optic tunable filter or improper heat dissipation. Spectrophotometer instability will generate unreliable spectra data, extra light reflections, and poor image quality. Galvanometer scanning instability yields poor image quality while microscope stage drift results in a sample going out of the plane of focus. With minor modifications, these tests may be applicable to other slide-based systems.

Animals↗
PubMed1997

Scanning laser ophthalmoscope imaging of fluorescein-labelled blood cells.

PURPOSE: To demonstrate the feasibility of a technique for the visualization by scanning laser ophthalmoscope (SLO) of fluorescein-labelled autologous leukocytes and platelets in retinal vessels. METHOD: Individual blood samples from rats and rabbits were centrifuged to isolate platelets and leukocytes, then passively labelled with fluorescein and reinjected into the same animal. An SLO was used to visualize and record cell displacement in the retinal circulation. Labelled platelets were analysed by flow cytometry. RESULTS: By SLO, platelets appeared as a heterogeneous particle flow, and individual leukocytes appearing as brighter spots could easily be traced. Flow cytometry showed that after labelling platelets were well individualized and their size was slightly increased. CONCLUSION: Circulating blood cells can be visualized in retinal vessels by a simple method consisting of passive labelling of autologous platelets and leukocytes by fluorescein. No platelet toxicity was detected. This method could be applied to the study of blood cell movement in human retinal vascular diseases.

Animals↗
PubMed2005

Effects of N- and C-terminal truncation of HP (2-20) from Helicobacter pylori ribosomal protein L1 (RPL1) on its anti-microbial activity.

HP (2-20) [derived from the N-terminal region of Helicobacter pylori Ribosomal Protein L1 (RPL1)], a 19-mer peptide, possesses broad-spectrum anti-microbial activity. As the N- (residues 2-3) and C-terminal (residues 14-20) residues can be deleted without affecting antimicrobial activity, we have now determined the minimum chain length necessary for the retention of antimicrobial activity, and its mode of action. The N- (residues 2-3) and C-terminal (residues 17-20) truncated fragments [HP (4-16)] induce increased antibiotic activity against several bacterial strains without hemolysis. Flow cytometric analysis, scanning electron microscopy and fluorescence confocal microscopy revealed that HP (4-16) acted rapidly on the plasma membranes of the fungal cells in a salt- and energy-independent manner.

Amino Acid Sequence↗
PubMed2002

MGr1-Ag is associated with multidrug-resistant phenotype of gastric cancer cells.

BACKGROUND: MGr1-antigen (Ag) was previously reported as an upregulated protein in multidrug-resistant (MDR) gastric cancer cells. The aim of this study was to characterize the role of MGr1-Ag in the multidrug resistance of gastric cancer cells. METHODS: Laser scanning confocal microscopy (LSCM), two-dimensional electrophoresis, and Western blot were used to detect MGr1-Ag in gastric cancer cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine the sensitivity of the MDR gastric cancer cells, SGC7901/VCR, to chemotherapeutic drugs. Adriamycin accumulation and retention in SGC7901/VCR cells were analyzed using flow cytometry. RESULTS: LSCM showed that MGr1-Ag localized mainly on the membrane and partly in the cytoplasm of SGC7901/VCR cells. Western blot showed that the expression level of MGr1-Ag in SGC7901/VCR cells was higher than that in its parental cells, SGC7901, and that the apparent molecular weight and isoelectric point of MGr1-Ag were 42 kDa and pH 4.8, respectively. After incubation with MGr1 antibody, SGC7901/VCR cells showed significantly decreased IC(50) values for adriamycin (from 0.887 +/- 0.081 mg/l to 0.607 +/- 0.084 mg/l; P, 0.05), vincristine (from 0.707 +/- 0.055 mg/l to 0.557 +/- 0.042 mg/l; P, 0.05), and 5-fluorouracil (from 4.367 +/- 0.407 mg/l to 2.630 +/- 0.644 mg/l; P, 0.05), as well as slightly increased IC(50) values for mitomycin (from 0.183 +/- 0.045 mg/l to 0.198 +/- 0.048 mg/l; P. 0.05). In addition, incubation with MGr1 significantly enhanced adriamycin accumulation and retention in SGC7901/VCR cells. CONCLUSION: Overexpression of MGr1-Ag is associated with the MDR phenotype of gastric cancer cells.

Antibodies, Monoclonal↗
PubMed1999

Expression of CD44 in human cumulus and mural granulosa cells of individual patients in in-vitro fertilization programmes.

CD44 is a polymorphic and polyfunctional transmembrane glycoprotein widely expressed in many types of cells. Here, the expression of this protein on human membrana granulosa was studied by two techniques. Using confocal laser scanning microscopy (CLSM) with the mouse monoclonal antibody to human CD44 (clone G44-26), cells immunoreactive for CD44 were observed in both cumulus and mural granulosa cell masses. On the other hand, using monoclonal antibody to human CD44v9, goat polyclonal antibody to human CD44v3-10 and the clone G44-26, no immunoreactivity for CD44v9 and/or CD44v3-10 was observed in either cell group by flow cytometry. In the flow cytometric analysis of 32 patients, the incidence of CD44 expression in cumulus cells (62.6+/-1.3%) was significantly higher than that in mural granulosa cells (38.5+/-3.2%) (P<0.0001). In the comparison of CD44 expression by flow cytometry according to the maturation of each cumulus-oocyte complex, the incidence of CD44 expression of cumulus cells was significantly higher in the mature group than in the immature group (P<0.05). In a flow cytometric analysis, patients with endometriosis showed a significantly lower incidence of CD44 expression in cumulus cells compared to the infertility of unknown origin group (P<0.05), and compared to both the male infertility group and the unknown origin group in mural granulosa cells (P<0.01). These findings suggest that the standard form of CD44 is expressed in human membrana granulosa with polarity and may play an important role in oocyte maturation.

Adult↗
PubMed2003

In vivo UVB irradiation induces clustering of Fas (CD95) on human epidermal cells.

In vitro studies with human cell lines have demonstrated that the death receptor Fas plays a role in ultraviolet (UV)-induced apoptosis. The purpose of the present study was to investigate the relation between Fas expression and apoptosis as well as clustering of Fas in human epidermis after a single dose of UVB irradiation. Normal healthy individuals were irradiated with three minimal erythema doses (MED) of UVB on forearm or buttock skin. Suction blisters from unirradiated and irradiated skin were raised, and Fas, FasL, and apoptosis of epidermal cells quantified by flow cytometry. Clustering of Fas was from skin biopsied. Soluble FasL in suction blister fluid was quantified by ELISA. Flow cytometric analysis demonstrated increased expression intensity of Fas after irradiation, with 1.6-,2.2- and 2.7-fold increased median expression at 24, 48 and 72 h after irradiation, respectively (n=4). Apoptosis was demonstrated by the TUNEL reaction, and the maximum of apoptotic cells was detected at 48 h after irradiation. Double-staining of Fas and TUNEL showed that apoptosis was restricted to the Fas-positive epidermal subpopulation, but there was no correlation between the intensities of Fas expression and TUNEL reaction. Median expression intensity of FasL-positive cells transiently decreased to 0.9- and 0.8-fold of the preirradiation respective level after 24 h and 48 h, respectively, and returned to the respective preirradiation level at 72 h after irradiation (n=4). Concentrations of soluble FasL in suction blister fluid from UVB-irradiated skin did not differ from those in unirradiated skin (n=5). Confocal laser scanning microscopy showed a rapid clustering of Fas within 30 min after irradiation. A simultaneous clustering of the adapter signalling protein FADD suggested that Fas clustering has a functional significance. Our results ar in accordance with previous findings from in vitro studies, and suggest that Fas is activated in vivo in human epidermis after UVB exposure.

Adult↗
PubMedDate not supplied

The methodology for studying coordinated calcium concentration changes in a pancreatic beta cell line.

Intracellular calcium concentration was imaged in beta Tc cells with the aid of Fluo-3 indicator and the Meridian ACAS 570 interactive laser cytometry. This cell line does not respond by an elevation in [Ca2+]in to increase in extracellular [glucose], but does respond to 10 microM forskolin. It was found that forskolin increases the mean [Ca2+]in and produces calcium spikes. Time series analysis was performed on individual pixels. Autocorrelation revealed that forskolin induces oscillation in [Ca2+]in. Cross-correlation analysis showed that all the intracellular pixels along the line scan are highly correlated, indicating that the increase in [Ca2+]in encompasses the entire cell.

Aniline Compounds↗
PubMed2003

Investigations of phagosomes, mitochondria, and acidic granules in human neutrophils using fluorescent probes.

The oxidative burst is frequently evaluated by the conversion of dihydrorhodamine 123 (DHR) to rhodamine 123 (R123) and hydroethidium (HE) to ethidium with the use of flow cytometry (FCM). Added R123 accumulates in mitochondria, but during phagocytosis R123 originating from DHR has been observed in neutrophil granules. The present study was designed to identify the site of reactive oxygen species (ROS) formation and the intracellular traffic of R123 in neutrophils by using mitochondrial membrane potential probes and the lysosomotropic probe LysoTracker Red, which have not previously been applied to neutrophils. Quiescent and phagocytosing human peripheral blood neutrophils were incubated with DHR, HE, R123, MitoTracker Green (MTG), MitoTracker Red (CMX-Ros), and LysoTracker Red alone and in all combinations of red and green probes, and studied by FCM and confocal laser scanning microscopy (CLSM). Phagosomes were filled with R123 originating from DHR. Phagocytosis also triggered the oxidative burst in oxidative response granules that differed from acidic granules. All the neutrophils stained with mitochondrial and lysosomotropic dyes. Added R123 and MTG selectively accumulated in mitochondria. Added R123, MTG, and DHR increased the fluorescence of CMX-Ros and LysoTracker Red. This is the first FCM and CLSM demonstration of ROS formation in phagosomes. A distinct subpopulation of neutrophil granules, termed oxidative response granules, also was identified. Neutrophil mitochondrial membrane potential may be evaluated by incubating the cells with R123 and MTG, but results with CMX-Ros should be interpreted with caution. HE and DHR seem to measure a common pathway in the oxidative burst. The simultaneous application of several probes for investigations of organelles carries the risk of probe interference.

Acids↗
PubMed1996

NKR-P1+ cells localize selectively in Rat 9L gliosarcomas but have reduced cytolytic function.

To better understand immune responses to brain tumors and to develop possible approaches for immunotherapy, we have investigated the leukocyte populations infiltrating the rat 9L gliosarcoma. By immunocytochem-ical analyses of the cells infiltrating the tumor, we observed a substantial number of cells expressing natural killer cell receptor protein 1 (NKR-P1), a marker expressed only on rat lymphocytes capable of non-MHC-restricted cytotoxicity. Previous investigations have determined the existence of three populations of NKR-P1+ lymphocytes in normal rats, including NKR-P1bright/T-cell receptor (TCR)-/CD3-/CD5- (approximately 5-15%), NKR-P1dim-/TCRalphabeta+/CD3+/CD5+ (approximately 1-5%), and NKR-P1dim/TCRgammadelta+/CD3+/CD5+ (approximately 0.5-2%). By one-parameter flow cytometry, it was determined that NKR-P1+ cells constituted 30-60% of the lymphocytes in 9L tumors. Among splenic lymphocytes or peripheral blood leukocytes, NKR-P1bright cells are 1.5-4.5 times more numerous than NKR-P1dim cells. In striking contrast, NKR-p1dim cells were 4-5 times more numerous than NKR-P1bright cells among lymphocytes isolated from 9L tumors. Using quantitative analyses of laser confocal microscopic scans, we determined that NKR-P1dim cells were approximately 4 times as numerous as NKR-P1bright cells in situ, confirming flow cytometric findings. By two-color now cytometric analyses, it was observed that approximately 5-10% of the cells were NKR-p1bright/CD5-/TCR-, a phenotype representative of NK cells. Also, approximately 11-25% of the cells were NKR-P1dim/CD5+/TCR+ cells, corresponding to the T-cell subset with non-MHC-restricted lytic function. In addition, we observed a cell population among 9L-derived lymphocytes with a NKR- p1dim/CD5-/TCR- phenotype (approximately 15-25%). Cells of this phenotype have not been reported previously, and most likely represent NK cells down-modulated for expression of NKR-P1. Alternatively, they might represent cells of unknown origin or cells down-modulated for expression of T-cell markers in the microenvironment of 9L tumors. We also compared the lytic capacity of NKR-P1+ populations derived from normal animals and from 9L gliosarcomas. In these experiments, it was determined that, although cells isolated from 9L tumors had some capacity to lyse tumor target cells, they were clearly less efficient than cells isolated from normal splenocytes. Cumulatively, these data suggest that there is selective localization of cells capable of mediating antitumor responses in 9L, but that tumor-associated factors may down-regulate their function and expression of NKR-P1.

Animals↗
PubMed2006

Mechanisms of co-modified liver-targeting liposomes as gene delivery carriers based on cellular uptake and antigens inhibition effect.

In order to deliver antisense oligonucleotides (asODN) into hepatocytes orientedly in the treatment of hepatitis B virus (HBV) infection, the liver-targeting cationic liposomes was developed as a gene carrier, which was co-modified with the ligand of the asialoglycoprotein receptor (ASGPR), beta-sitosterol-beta-d-glucoside (sito-G) and the nonionic surfactant, Brij 35. Flow cytometry (FCM) analysis and enzyme-linked immunosorbent assay (ELISA) showed that the asODN-encapsulating cationic liposomes exhibited high transfection efficiency and strong antigens inhibition effect in primary rat hepatocytes and HepG2.2.15 cells, respectively. With the help of several inhibitors acting on different steps during the targeting lipofection, the cellular uptake mechanisms of the co-modified liver-targeting cationic liposomes were investigated through antigens inhibition effect assay and confocal laser scanning microscopy (CLSM) analysis. The cellular uptake with high transfection efficiency seemed to involve both endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance ASGPR-mediated endocytosis, the nonionic surfactant Brij 35 seemed to be able to facilitate membrane fusion, and the co-modification resulted in the most efficient transfection but no enhanced cytotoxicity. These results suggested that the co-modified liver-targeting cationic liposomes would be a specific and effective carrier to transfer asODN into hepatocytes infected with HBV orientedly.

Androstadienes↗
PubMed2000

Relationship between actin microfilaments and plasma membrane changes during apoptosis of neoplastic cell lines in different culture conditions.

In this study we investigated the relationship between the reorganisation of actin cytoskeleton and the changes at cell surface level (i.e. PS exposure and blebbing) in two neoplastic cell lines during apoptosis: Chang liver cells (adherent culture) and promyelocytic HL-60 cells (suspension culture), treated with the podophyllotoxin derivative VP16. The morphological analysis, performed by means of conventional fluorescence microscopy and confocal laser scanning microscopy, on Chang cells showed that onset and progress of the two processes are synchronised. The initial disassembly of stress fibers was associated with the early PS exposure on the cell surface. Moreover, the accumulation of actin at cortical level appeared strongly associated with an intense labelling for Annexin V and, in some cases, especially in the areas of membrane blebbing. The double staining for actin and PS exposure, quantitatively analysed by flow cytometry in HL-60 cells after different treatment times, demonstrated that the decrease of Annexin V binding in the late stages of apoptosis is associated with the strong reduction of actin labelling probably also due to a proteolytic cleavage. These events were also partially related to variations of the functional state of mitochondria, by analysing cytofluorometrically the dissipation of the inner membrane potential (delta psi m).

Actin Cytoskeleton↗