Author investigated the deposition of immunoglobulins and complements in thc skin of 56 patients with 19 various dermatoses by direct immunofluorescent (DIF) staining. The biopsied specimens were quick-frozen(by dry ice-acetone) and stained with FITC conjugated antihuman immunoglobulin and complement after cutting in a cryostat at -20C~30C. (countinued...)
Complement System Proteins ; Dronabinol ; Fluorescein-5-isothiocyanate ; Humans ; Immunoglobulins ; Skin ; Skin Diseases*

Natural cytotoxicity mediated by natural killer (NK) cells is believed to play an important role in host anticancer defense mechanisms. The aim of this study is to compare the number of NK cells in patients with colorectal cancer and hemorrhoids, and before and after surgery in patients with colorectal cancer. Twenty colorectal cancer patients and twenty hemorrhoid ones were studied. Venous blood samples were obtained preoperatively, and on the 7th, and 14th postoperative days. Mononuclear cells were isolated over Ficoll-Hypaque gradients, and T cells, B cells, and NK cells were measured with CD3 FITC (T cell), CD 19 PE (B cell), and CD56 FITC (NK cell) antibody, The number of T cell (/mm3) was 1224, 1280, and 1125 at preoperative, 7th, and 14th postoperative day in hemorrhoid patients and 1195, 901, and 1060 in colorectal cancer patients respectively. The number of B cell (/mm3) was 243, 160, and 250 in hemorrhoid patients and 147, 78, and 113 in colorectal cancer patients. The number NK cell (/mm3) was 148, 156, and 143 in hemorrhoid patients and 129, 85, and 128 in colorectal cancer patients. There was no difference among Dukes stages in the number of NK cells. In conclusion, the number of NK cells was not changed in colorectal cancer patients compared with hemorrhoid ones. Major operation changed the number of NK cells in colorectal cancer patients.
B-Lymphocytes ; Colorectal Neoplasms* ; Defense Mechanisms ; Fluorescein-5-isothiocyanate ; Hemorrhoids ; Humans ; Killer Cells, Natural* ; T-Lymphocytes

AIMTo establish a simple and effective method for RBCs labeling and survival assays, and the qualities of rabbit RBCs preserved in GMA solution at 4 degrees C were verified.

METHODSThe bloods were taken through the ear arteries of the rabbits. The RBCs were labeled by fluorescein isothiocyanate (FITC), and were reinjected to the same rabbit through ear veins. The percentage of FITC labeled RBCs was assayed by FACS at a series of times after injection. The SAS software was employed to analyze the data and establish the regression equations. The 24-hour recovery and the half-life span of the labeled RBCs were calculated according to the equations.

RESULTSThe 24-hour recovery and the half-life span of the labeled RBCs in the control group were 93.76% +/- 5.40% and 22.50% +/- 4.37 days respectively, which was in agreement with the previous papers. The 24-hour recovery and the half-life span of the labeled RBCs in the GMA group were 89.13% +/- 7.10% and 11.41% +/- 1.63 days respectively, which was coincident with the infusion conditions.

CONCLUSIONCompared with other methods of RBCs labeling in vivo, FITC labeling was thought to be easier and cheaper to use, which could facilitate the analysis of the biological character of the labeled cells, and could be used to trace the fate of labeled cells.

Animals ; Blood Preservation ; methods ; Erythrocyte Aging ; physiology ; Erythrocyte Count ; Erythrocytes ; physiology ; Fluorescein-5-isothiocyanate ; Rabbits ; Software

The objective of this work is to evaluate the effect of polyamidoamine (PAMAM) dendrimers on electroosmotic flow (EOF) through skin. The effect of size and concentration of dendrimer was studied, using generation 1, 4 and 7 dendrimer (G1, G4 and G7, respectively). As a marker molecule for the direction and magnitude of EOF, a neutral molecule, acetoaminophen (AAP) was used. The visualization of dendrimer permeation into the current conducting pore (CCP) of skin was made using G4–fluorescein isothiocyanate (FITC) conjugate and confocal microscopy. Without dendrimer, anodal flux of AAP was much higher than cathodal or passive flux. When G1 dendrimer was added, anodal flux decreased, presumably due to the decrease in EOF by the association of G1 dendrimer with net negative charge in CCP. As the generation increased, larger decrease in anodal flux was observed, and the direction of EOF was reversed. Small amount of methanol used for the preparation of dendrimer solution also contributed to the decrease in anodal flux of AAP. Cross-sectional view perpendicular to the skin surface by confocal laser scanning microscope (CLSM) study showed that G4 dendrimer-FITC conjugate (G4-FITC) can penetrate into the viable epidermis and dermis under anodal current. The permeation route seemed to be localized on hair follicle region. These results suggest that PAMAM dendrimers can permeate into CCP and change the magnitude and direction of EOF. Overall, we obtained a better understanding on the mechanistic insights into the electroosmosis phenomena and its role on flux during iontophoresis.
Acetaminophen ; Dendrimers* ; Dermis ; Electroosmosis* ; Epidermis ; Fluorescein-5-isothiocyanate ; Hair Follicle ; Iontophoresis ; Methanol ; Microscopy, Confocal ; Skin*

In order to prepare angiopep-2 modified fluorescein isothiocyanate-labeled neurotoxin nanoparticles( ANG-NPs/FITCNT),emulsion/solvent evaporation method was used with m PEG-PLA and ANG-PEG-PLA( in proper proportions) as carriers and with FITC-NT as drug. With particle size and encapsulation efficiency as comprehensive indexes,the effects of different ultrasound power and ultrasound time combinations on the process were investigated. The in vitro release characteristics of nanoparticles in PBS buffer at p H 7. 4 and p H 6. 5 were investigated by dialysis method. The results indicated that the optimum process for preparing ANG-NPs/FITC-NT was as follows: ultrasonic power 90 W,ultrasonic time 30 s. In such optimal process,ANG-NPs/FITC-NT were well-shaped under the transmission electron microscope,with an average particle size of( 123. 9±0. 5) nm,Zeta potential of(-10. 5±0. 5) m V,encapsulation efficiency of( 68. 1±0. 4) %,and the drug loading of( 0. 82±0. 01) %. The in vitro drug release profiles of the nanoparticles in PBS buffer at p H 7. 4 and p H 6. 5 were both consistent with Ritger-Peppas equation,ln Q = 0. 508 8 lnt-2. 285 0,r = 0. 961 5( p H 7. 4) and ln Q= 0. 449 9 lnt-1. 855 3,r = 0. 970 3( p H 6. 5),respectively. The experiment results proved that the nanoparticles prepared by emulsion/solvent evaporation method had uniform particle size,high encapsulation efficiency and in vitro sustained release characteristic,which might be a potential carrier for NT intracerebral drug delivery.
Drug Carriers ; Fluorescein-5-isothiocyanate ; Nanoparticles ; Particle Size ; Peptides ; Polyethylene Glycols

BACKGROUND: Though elastic fibers are as important as collagen fibers in interpretation of the histopathologic findings, it is impossible to observe them on the hematoxylin & eosin (H&E) stained specimen. OBJECTIVE: Characterizing eosin fluorescence emitted by elastic fibers in H&E stained specimens. METHODS: Normal skin tissue sections were stained in 4 different ways (unstained, hematoxylin only, eosin only, H&E) and observed under a fluorescence microscope using a FITC filter set. Fluorescent findings of 30 H&E-stained specimens showing abnormal dermal findings were compared with bright field findings of Miller's elastic stained specimen. RESULTS: Strong eosin fluorescence was related to the differential binding property of eosin with elastic fibers. Hematoxylin stain quenched excessive eosin fluorescence from other tissue components and contributed to better contrast. Fluorescence microscopy of H&E-stained sections was found to be especially useful in observing mature elastic fibers in the reticular dermis. In 74% of the specimens, eosin fluorescence findings of elastic fibers in reticular dermis matched well with that of specimens with elastic fiber special stain. CONCLUSION: Analysis of skin elastic fibers by fluorescence microscopy is a useful and complementary method to reveal hidden elastic fibers in H&E-stained specimens.
Collagen ; Dermis ; Elastic Tissue ; Enzyme Multiplied Immunoassay Technique ; Eosine Yellowish-(YS) ; Fluorescein-5-isothiocyanate ; Fluorescence ; Hematoxylin ; Microscopy, Fluorescence ; Skin

To investigate the changing patterns of microfilament and microtubule arrangement and influence of myocardial cells and colchicines to microfilament and microtubule formation in cardiac endothelial cells the authors carried out indirect immunofluorescence stain for actin and tubulin with supernatant monoclonal antibodies. Secondary antibodies were IgG FITC conjugate. The results were summarized as follows. Fiberform reactions were stronger in the cells with many processes and spread cytoplasm and they became weaker after the endothelial cells formed monolayer. In the endothelial cells cocultured with myocardial cells the fiberform of the microtubule became less visible compared to control group but fiberform of the microtubule maintained strong intensity as endothelial cells formed monolayer. In the group treated with colchicines, there were no visible differences in microfilaments compared to control group but fiberform of microtubule revealed weaker intensity after colchicines treatment. The intensity of microtubule fiberform returned to control level after 2 days.
Actin Cytoskeleton ; Actins ; Antibodies ; Antibodies, Monoclonal* ; Cytoplasm ; Endothelial Cells* ; Fluorescein-5-isothiocyanate ; Fluorescent Antibody Technique, Indirect ; Immunoglobulin G ; Microtubules ; Tubulin

PURPOSE: Small size of recombinant scFv antibody has many advantages such as rapid blood clearances and improved targeting antibodies to tumor region. On the other hand owing to small size, number of amino group is insufficient in conjugation with chelator and fluorescence labeling. This study is to introduce poly lysine tag to the C-terminal end of scFv lym-1 sequence for fluorescence chelator conjugation. MATERIALS AND METHODS: Poly lysine scFv lym-1 gene, cloned into pET-22b (+) vector, was expressed in E. coli BL21 (DE3) strain. Antibody purification was performed with Ni-NTA column and then size exclusion column chromatography. Expression and purification levels of poly lysine tagged scFv lym-1 antibody were confirmed by western blot analysis. I-124, I-125, I-131 and Tc-99m were used for radiolabeling of purified poly lysine scFv lym-1. Flow cytometry analysis of FITC conjugated poly lysine scFv lym-1 was performed for confirmation of immunoreactivity of human Burkitt`s lymphoma cells. RESULTS: Poly lysine scFv lym-1 antibody was purified through two steps and identified as molecular weight of 48 KDa. Radiolabeling yields of I-124, I-125, I-131 and Tc-99m into poly lysine scFv lym-1 were >99%, >99%, >95% and >99%, respectively. Flow cytometry analysis of poly lysine scFv and scFv lym-1 was showed similar immunoreactivity to human Burkitt`s lymphoma cells. CONCLUSION: Poly lysine tag was useful for the sufficient number of amino groups to scFv lym-1 antibody for chelator conjugation with minimizing loss of immunoreactivity.
Antibodies ; Blotting, Western ; Chromatography ; Clone Cells ; Flow Cytometry ; Fluorescein-5-isothiocyanate ; Fluorescence ; Hand ; Humans ; Lymphoma ; Lysine ; Molecular Weight ; Sprains and Strains

PURPOSE: Numerous investigations have been conducted in order to determine the potential carcinogenic or chemopreventive activity of capsaicin. The aim of this study is to characterize the effects of capsaicin on colon cancer cells, and provide valuable information concerning the application of capsaicin in chemoprevention as well as for therapeutic purposes. METHODS: CoLo320DM and LoVo cells (human colon cancer cell line) were treated with capsaicin. In order to access cell viability and altered morphology, an MTT assay was performed and the cells were microscopically examined. Decreasing DNA staining was accessed by FACS. The cells were stained with FITC labeled annexin V and analyzed by FACS to detect cellular membrane alteration during apoptosis. The cells were stained with DiOC6(3) and Hydroethidine and analyzed by FACS in order to access ROS and dleta psi m. RESULTS: Capsaicin decreased cell viability in a dose-dependent manner. Capsaicin produced a cell morphology corresponding to the apoptotic features including cell shrinkage and chromatic condensation. Capsaicin treated cells induced a loss of nuclear DNA leading to hypoploidy in a dose-dependent manner. Cells were excluded by double staining with PI and FITC labeled annexin v and detected by FACS. We show that treatment of CoLo320DM, L0Vo cells with increasing concentrations of capsaicin parallel an increase in the percentage of red fluorescent cells (HE-->Eth) that reflect ROS hypergeneration and a decrease in the percentage of green fluorescent cells that reflect delta psi m disruption. CONCLUSION: These results clearly demonstrate that capsaicin-induced colon cancer cell death is apoptotic.
Annexin A5 ; Apoptosis* ; Capsaicin ; Cell Death ; Cell Line* ; Cell Survival ; Chemoprevention ; Colon* ; Colonic Neoplasms* ; DNA ; Fluorescein-5-isothiocyanate ; Humans* ; Membranes

OBJECTIVE: We investigated the effects on the cell cycle and p53 expression by the treatment of various concentrations of staurosporine to elucidate the molecular mechanism of staurosporine induced cell cycle arrest in MCF-7 cell line. METHODS: Various concentrations of staurosporine were treated in MCF-7 cells cultured with RPMI 1640 media. The harvested cells were fixed and permeabilized with 1% paraformaldehyde and absolute methanol. Then the cells were stained indirectly with anti-p53 primary antibody and FITC conjugated goat anti-mouse(GAM)-IgG secondary antibody. Sequentially DNA were stained with 0.1% RNase and PI solution. These stained cells were analyzed by the standard FACScan flow cytometer. The obtained results were analyzed further with WinList 3.0, and ModiFit LT software program. RESULTS: MCF-7 cells were arrested mostly in G1 phase of cell cycle at 5-10 nM of staurosporine, however, the cells were arrested in G2 phase at 20-100 nM of staurosporine. The expressions of p53 protein were higher in the MCF-7 cells treated with both concentrations of 10 nM and 100 nM of staurosporine compaired with the control cells. This suggests that the p53 may be involved in the mechanism of G1 and G2M arrest of cell cycle in MCF-7 cell. CONCLUSIONS: The points of arrest in cell cycle differred depending on the concentrations of staurosporine and these cell cycle arrests at G0G1 and G2M pahse were related with p53 protein expression. It suggested that these results could be extended to study for staurosporine to be usefull as a potential anti-tumor agent.
Cell Cycle Checkpoints ; Cell Cycle* ; DNA ; Fluorescein-5-isothiocyanate ; G1 Phase ; G2 Phase ; Goats ; MCF-7 Cells* ; Methanol ; Ribonucleases ; Staurosporine*