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PubMed1978

Differential dichrome staining of tissue culture monolayers: alternate dyes and possible mechanism.

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.

Cell Nucleus↗
PubMed1983

Visualization of Legionella pneumophila in paraffin sections using hexamine silver.

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 microns. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.

Humans↗
PubMed1996

Acidification and recovery results in nuclear accumulation of supravital dyes during interphase.

Recent studies using real time imaging demonstrated relative nuclear insulation for ion-size particles. We show here that acidification and recovery converted the insulated interphase nuclei of KB carcinoma and nontumorigenic Chang cells into intense nuclear accumulating states marked by sequestration of the exogenous supravital dyes neutral red, methylene blue, and brilliant cresyl blue. The phenomenon was not affected by Na(+)-free and HCO3(-)-free conditions nor by the presence of cationic and anionic antiport regulators of cytosolic pH. Cytological, microspectrophotometric, and flow cytometric evaluation of whole cell populations showed that the nuclear influx was abolished by omitting the pH recovery response, and by modulating the recovery response. The abolition of nuclear influx in the presence of the P-ATPase and Fzero-ATPase inhibitors, vanadyl(IV) ions and oligomycin, respectively, suggest that H(+)-translocating ATPase pumps are involved in regulating cytosolic acidification in Na(+)-free and HCO3-conditions vanadyl(IV) inhibited nuclear uptake of supravital dyes in a dose dependent manner. Nuclear uptake of dyes, however, was not affected by up to 1 mM of genistein even though tyrosine-specific phosphorylation and DNA synthesis were abolished. Upgradient nuclear influx involving proton pump is novel. KB cancer cells and nontumorigenic Chang cells had differential dye accumulations induced by acidification and recovery.

Cell Line↗
PubMed1986

Potentiation of cytotoxic effect of lymphotoxin by anti-cancer drugs and elevated temperatures.

The cytotoxic lymphokine, lymphotoxin (LT), has been shown to possess antitumor effect in vitro and in vivo. We examined the effect of the combination of partially purified LT with anti-cancer drugs and elevated temperatures on mouse transformed fibroblast cell line, L-929, and two human carcinoma of the cervix cell lines, HeLa and ME180. The cells were treated for 7 hr with Adriamycin, cisplatin, or bleomycin. These cells were then incubated for 24 hr in the presence of LT. At the end of the incubation period, cytotoxicity was measured by the neutral red dye uptake assay. There was 10- to 47-fold potentiation of cytotoxicity of LT on L-929 cells. The potentiation of cytotoxicity on human carcinoma of cervix cell lines ranged from 3- to 23-fold. L-929 cells and ME180 cells were incubated for 7 hr at 40 or 42 degrees C followed by 24 hr of incubation in the presence of LT. The elevated temperature treatment also enhanced (5- to 9-fold) the cytotoxic effect of LT. DNA, RNA, and protein syntheses of the ME180 cells was measured following incubation at 42 degrees C. It was observed that all three parameters were suppressed by incubation at this temperature. It was, therefore, possible that the repair of LT damaged cells was hampered by the elevated temperature treatment. It is suggested that LT may have a potential as an anti-tumor agent in combination with selected therapeutic drugs and hyperthermia.

Antineoplastic Agents↗
PubMed1990

Intraocular pressure following peritomy and wet-field coagulation.

The changes in intraocular pressure (IOP) following peritomy and wet-field coagulation of 10 rabbit eyes and 7 human cataractous eyes were investigated. The IOP difference before and after 180-degree and 360-degree wet-field coagulation on rabbit eyes showed an average increase of 11.0 +/- 5.8 mmHg and 25.8 +/- 7.1 mmHg(mean +/- SD), respectively (p less than 0.05, p less than 0.01). The IOP of both groups in rabbit eyes were elevated for 2 to 3 days postoperatively and then became normal by the 7th postoperative day. The IOP difference before and after 180-degree peritomy and wet-field coagulation in human cataractous eyes showed an average increase of 8.4 +/- 5.8 mmHg(mean +/- SD)(p less than 0.05). The outflow facility was poor immediately following wet-field coagulation but then improved gradually by means of tonography and concentration changes of anterior chamber neutral-red. Histologically, there was endothelial cell damage, congestion, and red blood cell extravasation of the episcleral and intrascleral vessels at one day after wet-field coagulation in the light microscopic findings. Recanalization of the episcleral and intrascleral capillaries was noted 3 days after wet-field coagulation and was completed 3 weeks after wet-field coagulation. There was poor plastic infusion through the trabecular spaces in the one-day postoperative group compared to the control group by scanning electron micrograph. In conclusion, we have to bear in mind the possibility of an IOP rise after wet-field coagulation following peritomy for cataract and retinal surgery.

Animals↗
PubMed2005

Effects of dendritic cells from cord blood CD34+ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice.

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in severe combined immunodeficiency (SCID) mice. METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocarcinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect. RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%, 47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80% vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11 d vs 7 d, P<0.01). CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.

Animals↗
PubMed2005

Nerve-pathways of acupoint Fengch'ih in rat by anterograde transport of HRP.

AIM: To study the nervous-pathways of Fengch'ih acupuncture by means of anterograde transport of aqueous solution of horseradish peroxidase (HRP). METHODS: Fifty Wistar rats were randomly divided into 1, 2, 3, 4, and 5 d groups, and every group had 10 animals. HRP (30% aqueous solution) was injected into a Fengch'ih. Serial, transverse or capital, 40 microm sections of the cervical spinal ganglia, cervical and thoracic spinal cord segment and brain were cut on a cryotome. Sections were incubated for HRP histochemistry according to the tetramethylbenzidine (TMB). Part of the sections were counterstained with neutral red. RESULTS: After 1 d of survival times, many labeled cell bodies were found in 1-4 cervical spinal ganglia, anterior horn of 1-4 cervical spinal cord, ventromedial division of facial nucleus, accessory facial nucleus ipsilaterally. With increasing survival times, the intensity of labeled cells were slightly decreased. CONCLUSION: Fengch'ih may bring into full play its effect by correlation of posterior ear branch of facial nerve and anterior branch of 2-3 cervical nerve with 1-4 cervical the anterior horn of the spinal cord, ventromedial division of facial nucleus, accessory facial nucleus.

Acupuncture Points↗
PubMed2006

Effect of NaCl and Helicobacter pylori vacuolating cytotoxin on cytokine expression and viability.

AIM: To determine whether Helicobacter pylori (H pylori) vacuolating cytotoxin (VacA) regulates release of pro-inflammatory cytokines (IL-1beta, IL-8, TNF-alpha, and IL-6) or alters gastric epithelial cell viability and to determine whether NaCl affects these VacA-induced changes. METHODS: Vacuolating activity was determined by measuring the uptake of neutral red into vacuoles of VacA-treated human gastric epithelial (AGS) cells. AGS cell viability was assessed by direct cell counting. Specific enzyme-linked immunosorbent assays (ELISA) and reverse transcriptase-polymerase chain reaction(RT-PCR) were performed to examine the effects of H pylori VacA and NaCl on cell pro-inflammatory cytokine production in AGS cells. Immunohistochemical staining of gastric tissue from Mongolian gerbils was used to confirm VacA-induced pro-inflammatory cytokine production and the effects of NaCl on this VacA-induced response. RESULTS: Addition of VacA alone reduced AGS cell viability (P < 0.05), and this reduction was enhanced by high doses of NaCl (P < 0.05). VacA alone induced expression of TNF-alpha, IL-8 and IL-1beta, while NaCl alone induced expression of TNF-alpha and IL-1beta. Changes in mRNA levels in the presence of both VacA and NaCl were more complicated. For the case of TNF-alpha, expression was dose-dependent on NaCl. IL-6 mRNA was not detected. However, low levels of IL-6 were detected by ELISA. Positive immunohistochemical staining of IL-1, IL-6, and TNF-alpha was found in gastric tissue of H pylori-infected gerbils fed with either a normal diet or a high salt diet. However, the staining of these three cytokines was stronger in H pylori-infected animals fed with a 5 g/kg NaCl diet. CONCLUSION: VacA decreases the viability of AGS cells, and this effect can be enhanced by NaCl. NaCl also affects the production of pro-inflammatory cytokines induced by VacA, suggesting that NaCl plays an important role in H pylori-induced gastric epithelial cell cytotoxicity.

Animals↗
PubMed2002

Antitumor activities of human dendritic cells derived from peripheral and cord blood.

AIM: To observe the biological specialization of human peripheral blood dendritic cells (DC) and cord blood derived DC and its effects on effector cells killing human hepatocarcinoma cell line BEL-7402. in vitro. METHODS: The DC biological characteristics were detected with immunohistochemical and MTT assay. Two antitumor experiment groups are divided: peripheral blood DC and cord blood DC groups. Peripheral blood DC groups used LAK cells as the effector cells and BEL-7402 as target cells, while cord blood DC groups used CTL induced by tumor antigen twice pulsed DC as effector cells and BEL-7402 as target cells, additional peripheral blood DC and cord blood DC are added to observe its stimulating activities to effector cells. The effector's cytotoxicity to tumor cells were detected with neutral red colorimetric assay at two effector/target ratios of 5:1 and 10:1. RESULTS: Peripheral blood DC and cord blood DC highly expressed HLA-ABC, HLA-DR, HLA-DQ, CD54 and S-100 protein. The stimulating activities to lymphocyte proliferation were compared between experimental groups (DC added) and control group (no DC added), in six experiment subgroups,the DC/lymphocyte ratio was sequentially 0.25:100, 0.5:100, 1:100, 2:100, 4:100 and 8:100.A values were sequentially 0.75396+/-0.009, 0.84916+/-0.010, 0.90894+/-0.012, 0.98371+/-0.007, 1.01299+/-0.006 and 1.20384+/-0.006 in peripheral blood DC groups and 0.77650+/-0.005, 0.83008+/-0.007, 0.92725+/-0.007, 1.05990+/-0.010, 1.15583+/-0.011, 1.22983+/-0.011 in cord blood DC groups. A value was 0.59517+/-0.005 in control group. The stimulating activities were higher in experimental groups than in control group (P<0.01), which were increased when the DC concentration was enlarged (P<0.01). Two differently derived DCs had the same phenotypes and similar stimulating activities (P<0.05). In peripheral blood DC groups, the cytotoxicity of the LD groups (experimental groups) and L groups (control group) was 58.16%+/-2.03% (5:1), 46.18%+/-2.25% (10:1) and 38.13%+/-1.29% (5:1) and 65.40%+/-1.56% (10:1) respectively; in cord blood DC groups, TD groups (experimental groups) and T groups (control groups) were 69.71%+/-2.33 % (5:1), 77.64%+/-1.94% (10:1) and 56.89%+/-1.82% (5:1) and 60.99%+/-1.42% (10:1) respectively.The cytotoxicity activities were enhanced with increased effector/target ratio (P<0.01). At the same effector/target ratio, the cytotoxicity of experimental groups were bigger than that of control groups (P<0.01). The cytotoxicity activities of cord blood DC groups were higher than that of peripheral blood DC groups (P<0.01). CONCLUSION: Peripheral blood DC and cord blood DC are mature DC in morphology and function, both can enhance the effector cell killing activities to hepatocarcinoma cells. DC pulsed with tumor antigen can induce higher specific CTL activity than unpulsed DC.

Antigen-Presenting Cells↗
PubMed2003

cagA and vacA genotype of Helicobacter pylori associated with gastric diseases in Xi'an area.

AIM: To establish stock of clinical Helicobacter pylori (H. pylori) isolates, to perform cagA and vacA typing of these isolates, to evaluate the relationship between genotypes of cagA and vacA and upper gastrointestinal diseases and to assess the association of vacA genotypes with presence of the pathogenicity marker-cagA. METHODS: Clinical H.pylori strains were isolated from the antrum of 259 patients in Clumbia agar. The isolated H.pylori strains were identified by histology, and16SrRNA PCR. CagA genotypes were detected by colony hybridization, the probe was derived from the cloned plasmid PcagA, and digested by EcoRI-HindIII and the isolated PcagA DNA fragment was radioactively labelled by the random priming method. vacA genes types (s,m)and subtypes (s1a, s1b, s2) were typed by PCR. Vacuolating toxin was detected with neutral red absorb test. The results were treated statistically by chi(2) test, t test, and rank sum test. RESULTS: A total of 192 clinical H.pylori strains were isolated and the stock of Helicobacter pylori was established. The total positive rate of cagA was 87 % in all gastric diseases, and 95 % in gastric cancer group. There was a difference between gastric cancer group and the other groups (P<0.05) except duodenal ulcer group. The expression of type s1 of vacA was more than type s2 (P<0.05), and, the expression of type m1 was equal to type m2. In gastric cancer group, there was a difference between s1a and s1b (P<0.05), and s1a was more than s1b. Vacuolating toxins were more in Xi'an area isolates. CONCLUSION: The cagA(+) vacA type s1 clinical isolates are more in Xi'an area, but this can not serve as an index to predict gastric cancer.

Antigens, Bacterial↗
PubMed1999

Cytotoxicity of dental resin monomers in the presence of S9 mix enzymes.

The purpose of the this study was to evaluate the cytotoxicity of dental resin monomers in the presence of a rat liver S9 mix containing cytochrome P 450 enzymes. JTC-12 cells derived from a monkey kidney were seeded on a 96-well multi-well-plate at 9 x 10(3) cells per well. After cultivation, the S9 mix was added to the wells as an S9 mix group (+S9), and PBS- was added to the other wells as a none-S9 mix group (-S9), then 7 different concentrations of various monomers were added to each well. All the specimens were cultured for another 24 hrs. The cell survival ratios (CSR) were calculated by using a neutral red cytotoxicity assay. CSR for 50 micrograms/mL of Bis-GMA/S9 mix was 92.6% while for none-S9 mix it was 6.6%. The values of CSR for UDMA, Bis-MPEPP, EGDMA, TEGDMA, DMAEM, 4-META and HEMA exhibited a reduction in cytotoxicity in the presence of the S9 mix. There were significant differences between +S9 and -S9 for respective monomers (p < 0.05). However, there were no significant differences between +S9 and -S9 for MMA (p < 0.05).

Animals↗
PubMed2004

Evidence that HSP70 gene expression may be useful for assessing the cytocompatibility of dental biomaterials.

In the current studies, we examined the possibility of using HSP70 gene regulation as a cytocompatibility test for dental biomaterials. For this reason, we assessed the effects of three metal salts, HgCl2, CuSO4 and NiCl2 on HSP70 gene expression in HeLa S3 cells using real-time Taqman quantitative PCR. Incubation of the cells for 4 h in medium containing HgCl2 (20 or 40 microM), CuSO4 (157, 313, 625 or 1250 microM) or NiCl2 (5000 and 10000 microM) significantly induced HSP70 mRNA. The real-time Taqman quantitative PCR was able to detect HSP70 mRNA induction at 4-fold lower concentrations of HgCl2 and 8-fold lower concentrations of CuSO4 than the Neutral Red cell viability assay. These results indicate that real-time Taqman quantitative PCR, in combination with the monitoring of cell viability, may be a valuable tool for distinguishing between specific HSP70 mRNA induction and cytocompatibility of metals in dental biomaterials.

Analysis of Variance↗
PubMed2003

Hyaluronan uptake by adult human skin fibroblasts in vitro.

Low and high molecular weight hyaluronan (HA) was added to adult human fibroblasts grown in monolayer to assess its influence on CD44 expression, its internalisation and effect on cell growth. CD44 expression on the surface of in vitro fibroblasts was not modified by different concentrations of FCS, whereas it was sensitive to cell cycle, being higher in the growing than in the resting phase. Independently from molecular weight, upon addition of exogenous HA (from 0.1 up to 1 mg/mL) to fibroblasts in the growing phase, a slight but constant decrease of the expression of CD44 on the surface of fibroblasts was observed; moreover, HA induced a rearrangement of CD44 into patches in close relationship with the terminal regions of stress fibers, which became thicker and more rigid after a few hours from the addition of HA to the medium. Fluorescent HA, added to the culture medium, rapidly attached to the plasma membrane and in less than two minutes was observed within cells, partly in association with its receptor CD44. By the contemporary use of neutral red, which accumulates into functional lysosomes, the great majority of internalised HA was found within lysosomes. HA receptor RHAMM-IHABP was rather homogeneously localised within the cytoplasm of normal growing fibroblasts. Upon addition of HA, the RHAMM-IHABP distribution became discontinuous around the nucleus. Addition of HA to fibroblasts induced a significant inhibition of cell growth, which was dependent on HA concentration and irrespective of HA molecular weight, at least in the ranges tested. Results show that extra-cellular HA is rapidly taken up by human dermal fibroblasts together with its CD44 receptor, and transported mostly to the lysosomes. Both low and high molecular weight HA induced down-regulation of cell proliferation, which would seem to be mediated by HA catabolism.

Adult↗
PubMed1991

Inhibition of pinocytosis by hygrolidin family antibiotics: possible correlation with their selective effects on oncogene-expressed cells.

A fermentation broth of Streptomyces sp. SIPI-A4-0044 inhibited in vitro growth of src or ras oncogene-expressed (onc+) cells more strongly than that of oncogene-unexpressed (onc-) counterparts. The active components were isolated and identified as hygrolidin family antibiotics (HGL). In mixed cultures consisting of onc+ and onc- cells, at an appropriate ratio, HGL showed selective toxicity to focus like structures of onc+ cells, leaving monolayer areas of onc- cells little damaged. HGL rapidly inhibited pinocytosis, or the influx of neutral red into the cells, at concentrations partially inhibitory to the cell growth. In contrast, HGL only slightly inhibited the influx of 2-deoxyglucose, nucleosides and leucine and the syntheses of DNA, RNA and protein even at high concentrations. Upon prolonged exposure to sublethal concentrations of HGL, onc- cells but not onc+ cells recovered pinocytotic activity and resumed growth.

Animals↗
PubMed2001

Comparison of three in vitro assay systems used for assessing cytotoxic effect of heavy metals on cultured human keratinocytes.

The cell viability assay using cultured cells is of great advantage to elucidate the biological effect of potentially toxic substances. Recently, a novel assay system, Tetracolor One cell proliferation assay (Seikagaku Co., Tokyo, Japan), has been developed. In this report, we compare the results of the Tetracolor One assay regarding the cytotoxic effect of three heavy metal salts on cultured adult keratinocytes to those of the neutral red dye uptake assay and the MTT eluted stain assay. In this study, these three methods showed almost similar results. Compared to the other two methods, however, the Tetracolor One assay, which requires only one-step procedure before spectrophotometric measurement, is easier to use, and errors in measurement, which may be produced through the multistep procedure, are much less in this assay. Therefore, we believe that the Tetracolor One assay system is useful for assessing the cytotoxic effect of heavy metals on cultured human keratinocytes.

Adult↗
PubMed1998

Occupational allergy to aldehydes in health care workers. Clinical observations. Experiments.

The incidence of allergy to aldehydes (formaldehyde, glutaraldehyde, glyoxal) was examined in 280 health care workers suffering from skin lesions. Allergy was diagnosed in 64 (22.8%) patients. The majority of them (85.9%) were sensitive only to 1 aldehyde. Formaldehyde caused allergy slightly more frequently (13.9%) than glutaraldehyde (12.4%). Only 5 (1.9%) patients were sensitive to glyoxal. The irritant effect of aldehydes to the rabbit eye and skin was tested by the Draize and OECD methods. Formaldehyde and glutaraldehyde showed stronger irritant effect than glyoxal. The sensitizing activity of aldehydes was also confirmed in guinea pigs (using the Maximization Test and the OECD methods). Formaldehyde showed the strongest and most persistent reactions. Significantly higher eosinophil and basophil counts were found in the blood samples of the sensitized guinea pigs. Cytotoxicity of glutaraldehyde and glyoxal was tested on mouse 3T3-L1 fibroblasts by the Neutral Red Uptake and MTT Reduction Assay. It was shown that both aldehydes were cytotoxic, and that the cytotoxic effect of glutaraldehyde was stronger than that of glyoxal.

Adult↗
PubMed1998

[Cytotoxic properties of pancreatic ribonuclease, modified by the surface active substance oxanol KD-6].

Pancreatic RNase modified by the surface active substance oxanole KD-6 (OxRNase) was studied in respect to its cytotoxic action on cells. The studies included in vitro and in vivo tests with intravital staining of the cells by neutral red and the 3H uridine label, as well as the test with the preparation action on fusion of lysosomes and phagosomes. It was shown that in all the tests the hydrophobised RNase had a higher cytotoxic action versus the native enzyme. The analysis of the experimental data suggested that the cytotoxicity of the hydrophobised RNase was due to its action on the cell membrane structures including the lysosome membranes.

Animals↗
PubMed1997

Effect of brucine on mouse nonspecific immune responses.

AIM: To evaluate the effect of brucine (Bru) i.p. at analgesic doses on the nonspecific immune responses in normal and cyclophosphamide (Cyc)-treated mice. METHODS: The clearance of charcoal particles, the immune organ weights, the white blood cell counts in peripheral blood, the phagocytosis to neutral red (NR) of PMO and its IL-1 production in vitro were tested. RESULTS: In normal mice, Bru slightly enhanced the clearance of charcoal particles, the phagocytosis of PMO, IL-1 production, the immune organ weights and the WBC counts (P > 0.05), whereas in Cyc-induced subnormal immunity model mice, Bru greatly enhanced these nonspecific immune responses (P < 0.05 or P < 0.01). The effects of Bru were most marked i.p. at 10 mg.kg-1 in vivo or 0.1-10 mg.L-1 in vitro. CONCLUSION: Bru i.p. at an analgesic dosage has dose- and function-dependent immunoregulatory effects.

Adjuvants, Immunologic↗