Particulate Matter 2.5 Causes Deficiency in Barrier Integrity in Human Nasal Epithelial Cells

PURPOSE: The effect of air pollution-related particulate matter (PM) on epithelial barrier function and tight junction (TJ) expression in human nasal mucosa has not been studied to date. This study therefore aimed to assess the direct impact of PM with an aerodynamic diameter less than 2.5 μm (PM2.5) on the barrier function and TJ molecular expression of human nasal epithelial cells. METHODS: Air-liquid interface cultures were established with epithelial cells derived from noninflammatory nasal mucosal tissue collected from patients undergoing paranasal sinus surgery. Confluent cultures were exposed to 50 or 100 µg/mL PM2.5 for up to 72 hours, and assessed for 1) epithelial barrier integrity as measured by transepithelial resistance (TER) and permeability of fluorescein isothiocyanate (FITC) 4 kDa; 2) expression of TJs using real-time quantitative polymerase chain reaction and immunofluorescence staining, and 3) proinflammatory cytokines by luminometric bead array or enzyme-linked immunosorbent assay. RESULTS: Compared to control medium, 50 and/or 100 µg/mL PM2.5-treatment 1) significantly decreased TER and increased FITC permeability, which could not be restored by budesonide pretreatment; 2) significantly decreased the expression of claudin-1 messenger RNA, claudin-1, occludin and ZO-1 protein; and 3) significantly increased production of the cytokines interleukin-8, TIMP metallopeptidase inhibitor 1 and thymic stromal lymphopoietin. CONCLUSIONS: Exposure to PM2.5 may lead to loss of barrier function in human nasal epithelium through decreased expression of TJ proteins and increased release of proinflammatory cytokines. These results suggest an important mechanism of susceptibility to rhinitis and rhinosinusitis in highly PM2.5-polluted areas.

Application of anoptomagnetic probe Gd-DO3A-EA-FITC in imaging and analyzing the brain interstitial space / 北京大学学报(医学版)

OBJECTIVE@#To investigate the application of the optical magnetic bimodal molecular probe Gd-DO3A-ethylthiouret-fluorescein isothiocyanate (Gd -DO3A-EA-FITC) in brain tissue imaging and brain interstitial space (ISS).@*METHODS@#In the study, 24 male SD rats were randomly divided into 3 groups, including magnetic probe group (n=6), optical probe group (n=6) and optical magnetic bimodal probe group (n=12), then the optical magnetic bimodal probe group was divided equally into magnetic probe subgroup (n=6) and optical probe subgroup (n=6). Referencing the brain stereotaxic atlas, the coronal globus pallidus as center level, the probes including gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA), fluorescein isothiocyanate (FITC) and Gd-DO3A-EA-FITC of 2 μL (10 mmol/L) were injected into the caudate nucleus respectively, magnetic resonance imaging (MRI) was performed in the magnetic probe group and magnetic probe subgroup to image the dynamic diffusion and distribution of the probes in the brain ISS, a self-developed brain ISS image processing system was used to measure the diffusion coefficient, clearance, volume fraction and half-time in these two groups. Laser scanning confocal microscope (LSCM) was performed in vitro in the optical probe group and optical probe subgroup for fluorescence imaging at the time points 2 hours after the injection of the probe, and the distribution in the oblique sagittal slice was compared with the result of the first two groups.@*RESULTS@#For the magnetic probe group and magnetic probe subgroup, there were the same imaging results between the probes of Gd-DTPA and Gd-DO3A-EA-FITC. The diffusion parameters of Gd-DTPA and Gd-DO3A-EA-FITC were as follows: the average diffusion coefficients [(3.31±0.11)×10-4 mm2/s vs. (3.37±0.15)×10-4 mm2/s, t=0.942, P=0.360], the clearance [(3.04±0.37) mmol/L vs. (2.90±0.51) mmol/L, t=0.640, P=0.531], the volume fractions (17.18%±0.14% vs. 17.31%±0.15%, t=1.961, P=0.068), the half-time [(86.58±3.31) min vs. (84.61±2.38) min, t=1.412, P=0.177], the diffusion areas [(23.25±0.68) mm2 vs. (22.71±1.00) mm2, t=1.100, P=0.297]. The statistical analysis of each brain was made by t test, and the diffusion parameters were not statistically significant. Moreover, for the optical probe group and optical probe subgroup, the diffusion area of Gd-DO3A-EA-FITC [(22.61±1.16) mm2] was slightly larger than that of FITC [(22.10±1.29) mm2], the statistical analysis of each brain was made by t test, and the diffusion parameters were not statistically significant (t=0.713, P=0.492).@*CONCLUSION@#Gd-DO3A-EA-FITC shows the same imaging results as the traditional GD-DTPA, and it can be used in measuring brain ISS.

Modulation of Electroosmotic Flow through Skin: Effect of Poly(Amidoamine) Dendrimers

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.

Effects of propranolol on oxygen-induced retinal neovascularization in mouse / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate whether propranolol application as collyrium or intraperitoneal (IP) injection can promote the recovery of oxygen-induced retinopathy (OIR).</p><p><b>METHOD</b>Thirty-six 7-day-old mice were divided into the following 6 groups: normal control, propranolol eye drops, propranolol IP injection, eye drops negative control, IP injection negative control, and pathological model with 6 mice in each. In a typical model of OIR, litters of mice pups with their nursing mothers were exposed to an infant incubator to high oxygen concentration (75 ± 5)% between postnatal day (PD) 7 and PD12, prior to returning to room air. Two routes of propranolol treatment were assessed from PD12 to PD17: IP injection and eye drop, with doses 2 mg/(kg·time), three times a day. Another three groups were given citric acid buffer eye drops, IP injection of citric acid buffer, and negative control were not treated with any drug. Neonatal mice fed in normal conditions served as normal control. Mice were sacrificed at PD17 to evaluate the morphological changes of retinal vessels by fluorescein isothiocyanate-dextran perfusion and retinal whole mount. The retinal neovascularization was evaluated by counting the number of nuclei of the endothelial cell breaking through the internal limiting membrane (ILM).</p><p><b>RESULT</b>Compared with the oxygen-exposed group, the branches of retinal vessels went normal with a less un-perfused area in the propranolol eye drops and propranolol IP injection groups [(38.9 ± 9.9)% and (5.6 ± 2.3)% vs. (16.2 ± 10.0)% and (2.2 ± 0.8)%, (25.9 ± 5.0)% and (2.1 ± 2.7)%, F=36.12 and 14.55, P both<0.001]. The number of nuclei of endothelial cells breaking through the ILM on the retinal cross-section in the propranolol eye drops group decreased (14.2 ± 5.1) per slide, which was less than that in the oxygen-exposed group (49.1 ± 8.9) per slide and the propranolol IP injection group (18.0 ± 5.9) per slide; it was also less than that in the eye drops negative control group (47.4 ± 8.1) per slide (F=187.60, P<0.05). Moreover, the number of nuclei of endothelial cells breaking through the ILM on the retinal cross-section in the propranolol IP injection group was less than that in the IP injection negative control group (49.9 ± 7.1) per slide (P<0.05).</p><p><b>CONCLUSION</b>Propranolol could effectively inhibit the formation of retinal neovascularization in mice; the eye drops was more effective than the IP injection.</p>

Study on delivery efficiency and cytotoxicity of Hela cells with mPEG-PLGA-BSA-FITC-NPs nanocarrier / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To construct and obtain ideal protein delivery vectors by researching the delivery efficiency and cytotoxicity to Hela cells using mPEG-PLGA-BSA-FITC-NPs.@*METHOD@#The mPEG-PLGA nanoparticle was obtained through surface modification of PLGA with PEG, and deliver BSA-FITC into Hela cells in vitro. The positive cells were counted by Laser scanning confocal microscopy and the survival rate of Hela cells was calculated by MTT assay at different time points.@*RESULT@#mPEG-PLGA-BSA-FITC-NPs shows the classic nanometer size, and the encapsulation efficiency reached 51. 2%. At the same time, the nanoparticles possess characteristics of slow release. By optimizing the delivery conditions, the highest efficiency of mPEG-PLGA-BSA-FITC-NPs was above 65.2%, and the cellular viability was about 85.7%.@*CONCLUSION@#mPEG-PLGA-BSA-FITC-NPs nanoparticles can successfully carry the target protein into cells as safe and effective as novel delivery materials of protein in vitro, and has shown slow release characteristics. The mPEG-PLGA-BSA-FITC-NPs provide ideal delivery vector for future application in clinical treatment of disease using nano-materials.

Stabilized thiomer PAA-Cys-6MNA / 药学学报

The aimed of this study was to prepare stabilized thiomers to overcome the poor stability character of traditional thiomers. Poly(acrylic acid)-cysteine (PAA-Cys) was synthesized by conjugating cysteine with poly(acrylic acid) and poly(acrylic acid)-cysteine-6-mercaptonicotinic acid (PAA-Cys-6MNA, stabilized thiomers) was synthesized by grafting a protecting group 6-mercaptonicotinic acid (6MNA) with PAA-Cys. The free thiol of PAA-Cys was determined by Ellmann's reagent method and the ratio of 6MNA coupled was determined by glutathione reduction method. The study of permeation enhancement and stabilized function was conducted by using Franz diffusion cell method, with fluorescein isothiocyanate dextran (FD4) used as model drug. The influence of polymers on tight junctions of Caco-2 cell monolayer was detected with laser scanning confocal fluorescence microscope. The results indicated that both PAA-Cys and PAA-Cys-6MNA could promote the permeation of FD4 across excised rat intestine, and the permeation function of PAA-Cys-6MNA was not influence by the pH of the storage environment and the oxidation of air after the protecting group 6MNA was grafted. The distribution of tight junction protein of Caco-2 cell monolayer F-actin was influenced after incubation with PAA-Cys and PAA-Cys-6MNA. In conclusion, stabilized thiomers (PAA-Cys-6MNA) maintained the permeation function compared with the traditional thiomers (PAA-Cys) and its stability was improved. The mechanism of the permeation enhancement function of the polymers might be related to their influence on tight junction relating proteins of cells.

Preparation of cell penetrating peptide TAT and cleavable PEGco-modified liposomes loaded with paclitaxel and its in vitro apoptosis assay / 药学学报

The preparation method, serum stability, efficiency of cellular uptake and apoptosis induction of the cell penetrating peptide TAT and cleavable PEG co-modified liposomes loaded with paclitaxel (C-TAT-Lipo) were investigated. The best preparation procedure was performed by orthogonal test based on single factor screening method. First, the paclitaxel (PTX)-loaded liposomes were prepared by filming-rehydration method, evaluated with entrapment efficiency and polydispersity index. The morphology of C-TAT-Lipo was characterized by transmission electron microscopy. Turbidity variations were monitored in the presence of fetal bovine serum (FBS) to evaluate the serum stability of the liposomes developed here. Next, the efficiency of cellular uptake of different Rho-PE-labeled liposomes on B16F1 cells in vitro was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry. The quantitative analysis of apoptosis induced by different PTX-loaded liposomes was performed by Annexin V-FITC/PI double staining. The optimal formulation was as follows: Chol : lipid: 1 : 8 (molar ratio); drug : lipid: 1 : 40 (mass ratio); lipid concentration: 3 mmol x L(-1); temperature of hydration: 25 degrees C. The mean size and polydispersity index of C-TAT-Lipo were about (97.97 +/- 3.68) nm and 0.196 +/- 0.037, the zeta potential was (-0.89 +/- 0.45) mV, the entrapment efficiency of paclitaxel was (90.16 +/- 1.53)%. The particle sizes did not exhibit significant variations in 50% FBS over 24 h at 37 degrees C. The efficiency of cellular uptake of the C-TAT-Lipo increased 1.40 fold following the cleavage of PEG. Apoptosis analysis showed 59.3% increase of the apoptosis and necrosis profile of C-TAT-Lipo after the detachment of PEG shells, which was markedly higher than that of N-TAT-LP with or without glutathione and SL, respectively. The results indicate that the C-TAT-Lipo is successfully prepared by filming-rehydration method and shows significant antitumor activities.

Study on oral absorption enhancers of astragalus polysaccharides / 中国中药杂志

Astragalus polysaccharides was lounded to 4-(2-aminoethylphenol), followed by labeling the APS-Tyr with fluorescein-5-isothiocyanate (FITC) at the secondary amino group. The absorption enhancement effects of low molecular weight chitosan and protamine on astragalus polysaccharides were evaluated via Caco-2 cell culture model. The results show that the fluorecent labeling compound has good stability and high sensitivity. On the other hand low molecular weight chitosan and protamine also can promoted absorption of the astragalus polysaccharides without any cytotoxity, and the absorption increase was more significant with increasing the amount of low molecular weight chitosan and protamine. At the same time, the low molecular weight chitosan has slightly better effect. The transepithelial electric resistance (TEER) of Caco-2 cells show that absorption enhancers could improve its membrane transport permeability by opening tight junctions between cells and increasing the cell membrane fluidity.

Effects of endothelial microvesicles induced by A23187 on H9c2 cardiomyocytes / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effects of endothelial microvesicles (EMVs) induced by calcium ionophore A23187 on H9c2 cardiomyocytes.</p><p><b>METHODS</b>Human umbilical vein endothelial cells (HUVECs) were treated with 10 micromol/L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. EMVs were characterized using 1 and 2 microm latex beads and anti-PE-CD144 antibody by flow cytometry. For functional research, EMVs at different concentrations were cocultured with H9c2 cardiomyocytes for 6 h. Cell viability of H9c2 cells and the activity of LDH leaked from H9c2 cells were tested by colorimetry. Moreover, apoptosis of H9c2 cells was observed through Hoechst 33258 staining and tested by FITC-Annexin V/PI double staining.</p><p><b>RESULTS</b>EMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. We identified the membrane vesicles (< 1 microm) induced by A23187 were CD144 positive. In addition, the EMVs could significantly reduce the viability of H9c2 cells, and increase LDH leakage from H9c2 cells in a dose dependent manner (P < 0.05). Condensed nuclei could be observed with the increasing concentrations of EMVs through Hoechst 33258 staining. Furthermore, increased apoptosis rates of H9c2 cells could be assessed through FITC-Annexin V/PI double staining by flow cytometry.</p><p><b>CONCLUSION</b>Microvesicles could be released from HUVECs after induced by A23187 through calcium influx, and these EMVs exerted a pro-apoptotic effect on H9c2 cells by induction of apoptosis.</p>

Gambogic Acid Induced Apoptosis through Activation of Caspase-dependent Pathway in Aortic Smooth Muscle Cells / 체질인류학회지

Gambogic acid (GA) has powerful apoptotic actions. The authors investigated whether GA has apoptotic effects on aortic smooth muscle cells, and compared its potency with that of simvastatin. Smooth muscle cells were isolated from the aortas of Sprague-Dawley rats (4-6 week). Cell purities were confirmed by IF staining using alpha-smooth muscle actin antibody. The IC50 values for cell death by GA and simvastatin were determined using a MTT assay, and the apoptotic effects of 1 microM GA or 30 microM simvastatin (concentrations correspond to IC50 values) were determined after 24 h of treatment using live cell images and by FITC annexin-V and propidium iodide double-staining. In addition, western blotting was used to evaluate apoptosis by quantifying reductions in the expression levels of the PARP and procaspase-3 as well as cleavages of PARP and procaspase-3 after treatment with 1 microM GA or 30 microM simvastatin. The IC50 of GA (1 microM) was lower than that of simvastatin (30 microM). Cell numbers were markedly reduced by both drugs in live cell images. GA (1 microM) produced a higher level of apoptosis than 30 microM simvastatin (26.4+/-2.37% vs. 8.3+/-1.54%, respectively; P<0.05, n=3) by FITC annexin-V & PI double-staining. In addition, 1 microM GA reduced the expressions of PARP, procaspase-3, and Mcl-1 in cells, whereas 30 microM simvastatin did not. Pretreatment with z-VAD-fmk attenuated GA-induced apoptosis and the cleavages of PARP and procaspase-3. The decreased level of Mcl-1 protein induced by GA treatment was recovered by z-VAD-fmk. These results indicate that GA-induced apoptosis was mediated by a caspase-dependent pathway.

Berberine inhibits norepinephrine-induced apoptosis in neonatal rat cardiomyocytes via inhibiting ROS-TNF-α-caspase signaling pathway / 中国结合医学杂志

<p><b>OBJECTIVE</b>To determine the effect of berberine (Ber) on norepinephrine (NE)-induced apoptosis in neonatal rat cardiomyocytes.</p><p><b>METHODS</b>The cultured neonatal rat cardiomyocytes were treated with NE in the presence or absence of Ber. The activity of lactate dehydrogenase (LDH) in the culture medium was examined, and apoptosis of cardiomyocytes was assessed by Hoechst 33258, isothiocyanate (FITC)-conjugated annexin-V, and propidine iodide (PI) staining. In addition, the activities of caspases-2 and-3 were measured by a fluorescent assay kit. The level of secreted tumor necrosis factor α (TNF-α) and production of intracellular reactive oxygen species (ROS) were also determined.</p><p><b>RESULTS</b>NE at a concentration of 50 μ mol/L induced an obvious increase in the activity of LDH in the culture medium (P<0.05), which was inhibited by coincubation with 0.5, 1.0, or 2.0 μ mol/L Ber (P<0.05). Ber also significantly attenuated NE-induced apoptosis in a dose-dependent manner (P<0.01). Moreover, Ber at a dose of 2 μ mol/L markedly decreased the ROS and TNF-α productions (P <0.05) and inhibited the activation of caspases-2 and -3 in cardiomyocytes exposed to NE (P<0.05)h.</p><p><b>CONCLUSION</b>The present study suggested that Ber could reduce NE-induced apoptosis in neonatal rat cardiomyocytes through inhibiting the ROS-TNF-α-caspase signaling pathway.</p>

Chlorogenic Acid Decreases Retinal Vascular Hyperpermeability in Diabetic Rat Model

To evaluate the effect of chlorogenic acid (CGA), a polyphenol abundant in coffee, on retinal vascular leakage in the rat model of diabetic retinopathy, Sprague-Dawley rats were divided into four groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with 10 and 20 mg/kg chlorogenic acid intraperitoneally daily for 14 days, respectively. Blood-retinal barrier (BRB) breakdown was evaluated using FITC-dextran. Vascular endothelial growth factor (VEGF) distribution and expression level was evaluated with immunohistochemistry and Western blot analysis. Expression of tight junction proteins, occludin and claudin-5, and zonula occludens protein, ZO-1 was also evaluated with immunohistochemistry and Western blot analysis. BRB breakdown and increased vascular leakage was found in diabetic rats, with increased VEGF expression and down-regulation of occludin, claudin-5, and ZO-1. CGA treatment effectively preserved the expression of occludin, and decreased VEGF levels, leading to less BRB breakdown and less vascular leakage. CGA may have a preventive role in BRB breakdown in diabetic retinopathy by preserving tight junction protein levels and low VEGF levels.

Preparation and initial application of a monoclonal antibody specific for a newly discovered conserved linear epitope of rabies virus nucleoprotein / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test.</p><p><b>METHODS</b>Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate (FITC) after purification and used in fluorescent antibody test (FAT).</p><p><b>RESULTS</b>Two positive hybridoma cell lines, RVNP-mAb1-CL and RVNP-mAb2-CL, were obtained. RVNP- mAb1-CL produced a higher concentration of monoclonal antibody RVNP-mAb1 in Balb/c ascites. FITC-labeled RVNP-mAb1 showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line (BSR).</p><p><b>CONCLUSION</b>FITC-labeled RVNP-mAb1 has potential application for laboratory diagnosis of rabies.</p>

Single-color Multitarget Flow Cytometry Using Monoclonal Antibodies Labeled with Different Intensities of the Same Fluorochrome

BACKGROUND: We developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay. METHODS: We selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples. RESULTS: CV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein alpha-isothyocyanate (FITC)dim+CD4-FITCbright and with CD19-FITCdim+CD3-FITCbright showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-Thelper cells, and Thelper cells; r2=0.87, 0.97, 0.97, and 0.98, respectively; P or =0.98, 0.99, 0.99, and 0.99, respectively; P<0.05). CONCLUSIONS: The multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.

Synergistic Effect of Parthenolide in Combination with 5-Fluorouracil in SW480 Cells

BACKGROUND/AIMS: Parthenolide (PT) is responsible for the bioactivities of Feverfew. Besides its potent anti-inflammatory effect, this compound has recently been reported to induce apoptosis in cancer cells. Unfortunately, many of the therapies that use 5-fluorouracil (5-FU) alone or in combination with other agents are likely to become ineffective due to drug resistance. In the present study, we investigate the antitumor effect of PT combined with 5-FU on colorectal cancer cells. METHODS: SW480 cell was employed as a representative of human colorectal carcinoma (CRC) cells. We performed MTT, annexin-V assay, and Hoechst 33258 staining to measure the synergistic effect. Western blotting was used to demonstrate apoptotic pathway. RESULTS: Our result demonstrated that PT inhibited the viability of colorectal cancer cells and had synergistic anti-proliferation in combination with 5-FU. After combined treatment of 5-FU and PT, enhanced apoptotic cell death is observed using annexin-V FITC assay and it was revealed by the condensed chromatin and fragmented DNA. Compared with 5-FU or PT alone, the apoptosis of colorectal cancer cells treated with PT and 5-FU enhanced the activation of caspase-8, caspase-3. CONCLUSIONS: Combined treatment with PT may offer an efficacious strategy to overcome 5-FU resistance in certain CRC cells.

In vivo distribution of FITC-labeled boanmycin hydrochloride in situ gel / 药学学报

This study is to evaluate the sustained-release effect of the thermosensitive in situ gel for injection of boanmycin hydrochloride (BAM) by bioluminescence imaging in nude mice. BAM was labeled with fluorescein isothiocyanate (FITC). The FITC-labeled BAM (FITC-BAM) was purified by dialysis and Sephadex G25 gel column, and then was identified by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF). The model of experimental hepatoma HepG-2 nude mice was established, and the optical imaging system was applied to evaluate the distribution of FITC-BAM in vivo. Results of MALDI-TOF proved that the major molecular ratio of BAM : FITC was 1 : 1 or 1 : 2. Bioluminescence imaging showed that the diffusion of FITC-BAM in situ gel group was significantly delayed compared with the negative control group. This study demonstrated that the thermosensitive in situ gel can effectively delay the release of boanmycin hydrochloride, and extend the retention time in vivo.

In vitro study on gastrointestinal absorption of FITC labeled pilose antler protein extraction / 药学学报

An in vitro detection method of the gastrointestinal absorption of Pilose Antler protein was established for mixed protein activity. Five bands of protein with molecular weight of 17.8-160 kD derived from the Pilose Antler were extracted and sufficiently labeled with FITC (FITC-PE). The stability and variation of FITC-PE in gastrointestinal circumstances were detected by native polyacrylamide gel electrophoresis and confocal laser scanning microscope. Results showed that the main component of FITC-PE kept invariant after being reacted with artificial gastric fluid and artificial intestinal fluid. The fluorescence signal was detected 20 min after administration in the valgus intestinal purse experiment, and three kinds of protein, with molecular weight of 45, 25, and 17.8 kD, were detected in the mixture of absorbent protein. The research laid the foundation for the further in vivo study of Pilose Antler protein. Meanwhile, it would be an in vitro screening method for the absorption, distribution and metabolism of mixed protein from traditional Chinese medicine.

Comparative Quantitative Analysis of Cluster of Differentiation 45 Antigen Expression on Lymphocyte Subsets / 대한진단검사의학회지

BACKGROUND: Since the recent introduction of radioimmunotherapy (RIT) using antibodies against cluster of differentiation (CD) 45 for the treatment of lymphoma, the clinical significance of the CD45 antigen has been increasing steadily. Here, we analyzed CD45 expression on lymphocyte subsets using flow cytometry in order to predict the susceptibility of normal lymphocytes to RIT. METHODS: Peripheral blood specimens were collected from 14 healthy individuals aged 25-54 yr. The mean fluorescence intensity (MFI) of the cell surface antigens was measured using a FACSCanto II system (Becton Dickinson Bioscience, USA). MFI values were converted into antibody binding capacity values using a Quantum Simply Cellular microbead kit (Bangs Laboratories, Inc., USA). RESULTS: Among the lymphocyte subsets, the expression of CD45 was the highest (725,368+/-42,763) on natural killer T (NKT) cells, 674,030+/-48,187 on cytotoxic/suppressor T cells, 588,750+/-48,090 on natural killer (NK) cells, 580,211+/-29,168 on helper T (Th) cells, and 499,436+/-21,737 on B cells. The Th cells and NK cells expressed a similar level of CD45 (P=0.502). Forward scatter was the highest in NKT cells (P<0.05), whereas side scatter differed significantly between each of the lymphocyte subsets (P<0.05). CD3 expression was highest in the Th and NKT cells. CONCLUSIONS: NKT cells express the highest levels of CD45 antigen. Therefore, this lymphocyte subset would be most profoundly affected by RIT or pretargeted RIT. The monitoring of this lymphocyte subset during and after RIT should prove helpful.

Comparative Quantitative Analysis of Cluster of Differentiation 45 Antigen Expression on Lymphocyte Subsets / 대한진단검사의학회지

BACKGROUND: Since the recent introduction of radioimmunotherapy (RIT) using antibodies against cluster of differentiation (CD) 45 for the treatment of lymphoma, the clinical significance of the CD45 antigen has been increasing steadily. Here, we analyzed CD45 expression on lymphocyte subsets using flow cytometry in order to predict the susceptibility of normal lymphocytes to RIT. METHODS: Peripheral blood specimens were collected from 14 healthy individuals aged 25-54 yr. The mean fluorescence intensity (MFI) of the cell surface antigens was measured using a FACSCanto II system (Becton Dickinson Bioscience, USA). MFI values were converted into antibody binding capacity values using a Quantum Simply Cellular microbead kit (Bangs Laboratories, Inc., USA). RESULTS: Among the lymphocyte subsets, the expression of CD45 was the highest (725,368+/-42,763) on natural killer T (NKT) cells, 674,030+/-48,187 on cytotoxic/suppressor T cells, 588,750+/-48,090 on natural killer (NK) cells, 580,211+/-29,168 on helper T (Th) cells, and 499,436+/-21,737 on B cells. The Th cells and NK cells expressed a similar level of CD45 (P=0.502). Forward scatter was the highest in NKT cells (P<0.05), whereas side scatter differed significantly between each of the lymphocyte subsets (P<0.05). CD3 expression was highest in the Th and NKT cells. CONCLUSIONS: NKT cells express the highest levels of CD45 antigen. Therefore, this lymphocyte subset would be most profoundly affected by RIT or pretargeted RIT. The monitoring of this lymphocyte subset during and after RIT should prove helpful.

Characterizing Cutaneous Elastic Fibers by Eosin Fluorescence Detected by Fluorescence Microscopy

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.