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PubMedDate not supplied

Anti-cancer effect of adenovirus p53 on human cervical cancer cell growth in vitro and in vivo.

To evaluate anti-tumor effects of recombinant adenovirus p53, time-course p53, E6 expression, and cell growth inhibition were investigated in vitro and in vivo using cervical cancer cell lines such as CaSki, SiHa, HeLa, HeLaS3, C33A, and HT3. The cell growth inhibition was studied via cell count assay, MTT assay and neutral red assay. After transfecting AdCMVp53 into SiHa cells-xenografted nude mice, the transduction efficiency and anti-tumor effect were investigated for a month. The results showed that adenoviral p53 expression induced significant growth suppression on the cancer cells, in which E6 transcript was strongly repressed, and that the expression of p53 and E6 were remarkably dependent on each cell type. The transduction efficiency was highly maintained in vivo as well as in vitro, and the size of tumor was remarkably decreased in comparison with AdCMVLacZ control. The results suggest that the adenovirus-mediated p53 gene transfection was done very effectively in vitro and in vivo experiment, and the cell growth was suppressed via p53-dependent apoptotic cell death, and that the anti-tumor effect could be related to E6 and p53 expression pattern.

Adenoviruses, Human↗
PubMed1990

Comparative evaluation on mouse nasal immunogenicity of arylmethane-, xanthene-, quinone-imine-, and acridine-dye-inactivated Sendai virus vaccines.

Twenty-seven kinds of organic dye-inactivated Sendai virus vaccines were prepared by treatment in dark at 23 C for 2 months or more, and selected with the high HA titers as a guide. Their nasal immunogenicities were examined in mice by contact infection and immunofluorescent method, and the relative merits of the dye-inactivants were determined. The strongest protection was elicited with acriflavine-, auramine O-, eosin Y-, neutral red-, night blue-, patent blue V-, thymol blue-, uranin-, and xylene cyanol FF-treated vaccines. Middling protective efficacy was induced by use of erio green B-, malachite green-, methyl green-, proflavine-, pyronin B-, and thionin-inactivated vaccines. Dye-inactivated vaccines that resulted in the weakest protection were Bindschedler's green-, bromothymol blue-, erythrosin B-, ethyl violet-, gallein-, light green SF yellowish-, methyl violet-, new methylene blue N-, phenol red-, rhodamine 6G-, spirit blue- and victoria blue B-treated ones. Serum HI titers developed by nasal vaccination were variable, and rose still more in most vaccinated groups postexposure. Elicitation of the most effective nasal immunogenicity in dye-inactivated vaccines appeared to depend on selective modification of capsid protein or ribose in viral core with dyes possessing definite functions, despite the different molecular structures.

Animals↗
PubMed2001

Examination of in vitro chemosensitivity test using collagen gel droplet culture method with colorimetric endpoint quantification.

To develop a simpler method of performing the collagen gel droplet-embedded culture drug sensitivity test (CD-DST), we examined the introduction of colorimetric quantitative determination of images for evaluation of anticancer effect against cancer cells alone in the presence of fibroblasts, based on differences in proliferative morphology and stainability with neutral red of cells within collagen gel drops determined using a video-microscope and NIH Image software. In examinations using a human cancer cell line and a fibroblast cell line, a high degree of linearity between number of cancer cells and image-optical density was found within the range of 10(2) - 10( 6) cells / droplet (r (2) = 0.933). Using NIH Image, fibroblast cells could be eliminated at a cut-off value of 128, and an immunocytochemical method demonstrated that the cells eliminated from the image were indeed fibroblasts, and those remaining were cancer cells. CD-DST was carried out with mixtures of cancer cells with fibroblasts at various ratios, and the feasibility of evaluating anticancer activity in cancer cells alone with no effect of fibroblasts at any mixing ratio was confirmed. In addition, for CD-DST of primary cell cultures of human lung cancers collected at the time of surgery, a high correlation between results obtained with the volume supplementation method, a current cell quantification method, and those with the imaging colorimetric quantification method was obtained (r = 0.933). These results indicate that introduction of imaging colorimetric quantification utilizing NIH Image makes CD-DST a quick and simple method that should be highly useful for clinical chemosensitivity testing using primary cell cultures of human cancers.

Antineoplastic Agents↗
PubMed1994

Quantitative in vitro assessment of N-alkyl sulphate-induced cytotoxicity in human keratinocytes (HaCaT). Comparison with in vivo human irritation tests.

A spontaneously immortalized human keratinocyte line, HaCaT, was used as an in vitro model to predict the cutaneous irritation of anionic surfactants. For this purpose, a number of sodium salts of N-alkyl sulphates with hydrocarbon chain lengths varying between C8 and C16 were studied for possible cytotoxic effects. The endpoints used to assess toxicity were uptake of the vital dye neutral red (NR) and cell morphology criteria 24 h after dosing. A linear proportionality between keratinocyte number and NR uptake was established. All tested surfactants had cytotoxic effects as demonstrated by a decreased NR uptake, which showed a clear dose-response relationship. Concentrations resulting in 50% inhibition of NR uptake (IC-50) ranged from 0.15 mmol (sodium lauryl sulphate, C12) to 1.23 mmol (sodium octyl sulphate, C8). The in vitro cytotoxicity data were highly reproducible when the test was repeated after several weeks. The cytotoxicity data from these assays were compared with the irritant responses (as evaluated by measurement of erythema and transepidermal water loss) obtained after 24 h application of the same compounds (300 microliters of 20 mmol aqueous solution) to the volar forearm of human volunteers. There were significant linear correlations between the IC-50 values and both barrier damage (transepidermal water loss) and erythema (as evaluated by skin colour reflectance measurements). For the test substances, however, the sensitivity of the in vitro system was between 10 and 100 times higher than that observed in human skin in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1995

Acyclovir-resistant chronic cutaneous herpes simplex in Wiskott-Aldrich syndrome.

A 28-year-old man with Wiskott-Aldrich syndrome presented with ulcerative-proliferative lesions on his face from which herpes simplex type 1 (HSV-1) was isolated. He was initially treated with 10 mg/kg of acyclovir (Zovirax) intravenously every 8 h, but his skin lesions worsened. Clinical resistance to acyclovir was suspected, and therapy with this drug was intensified. The dosage of acyclovir was increased to 45 mg/kg, administered by continuous infusion, and the lesions subsequently resolved. The strain of HSV recovered from the patient showed acyclovir-resistance in vitro, using the colorimetric method with neutral red. Herpes simplex virus resistance to acyclovir is rare. It is more common in immunocompromised patients if subtherapeutic doses are administered in the treatment of chronic persistent forms of infection. Whenever clinical resistance to acyclovir is suspected, the dosage should be increased to 2 mg/kg per h administered via an infusion pump. If no improvement is observed in the patient's condition with this regimen, a phosphorylated medication whose mechanism of action is not dependent on viral thymidine kinase, such as foscarnet (phosphonoformic acid), should be substituted.

Acyclovir↗
PubMed1975

Quantitation of subclasses of mononuclear cells in normal human blood by membrane receptor studies.

A technique for quantitating mononuclear cells expressing E, EA(IgG), EAC receptors, immunoglobulin carrying cells and phagocytic cells in normal human venous blood is described; normal values for each of these classes of mononuclear cell are established. The effects of different methods of cell preparation have been considered; these were found not to have a significant effect on the final values. In addition, we describe a second category of 'fluorescent non-capping' cells and show that these are numerically equivalent to cells which ingest neutral red.

Adult↗
PubMed2006

Not only insulin stimulates mitochondriogenesis in muscle cells, but mitochondria are also essential for insulin-mediated myogenesis.

Viability and myogenesis from C2C12 muscle cells and L6 rat myoblasts were dose-dependently stimulated by insulin. The metabolic inhibitors of phosphatidyl-inositol-3-kinase (PI-3K, LY294002) and of MAPKK/ERK kinase (MEK, PD98059) differently affected insulin-stimulated myogenesis of the cells. After LY294002 and PD98059 treatment, viability deteriorated and apparently an additive effect of both metabolic inhibitors was observed, irrespective of the method of measurement (neutral red or MTT assay). These inhibitors were antagonistic in myogenesis. Our results confirm that insulin regulates cell viability by at least two distinct pathways, namely by PI-3K- and MEK-dependent signalling cascades. Both pathways are agonistic in cell viability, whereas PI-3K rather than MEK supports insulin-mediated myogenicity. Accordingly, inhibition of insulin action by LY294002, but not PD98059, was accompanied with a reduced level of Ser473-phosphorylated Akt with additional loss of myogenin protein. Besides, repression of insulin signalling by either PI-3K or MEK inhibitor diminished expression of selected subunits of the mitochondrial oxidative phosphorylation enzymes (OXPHOS). In turn, insulin raised and accelerated protein expression of subunits I and IV of mitochondrial cytochrome-c oxidase (COX). In addition, the level of myogenin, the molecular marker of terminal and general muscle differentiation indices decreased if selected OXPHOS enzymes were individually blocked by rotenone, myxothiazol or oligomycin. Summing up, our results pointed to mitochondria as an essential organelle for insulin-dependent myogenesis. Insulin positively affects mitochondrial function by induction of OXPHOS enzymes, which provide energy indispensable for the anabolic effect of insulin.

Animals↗
PubMed1992

Activated T-lymphocytes induce growth inhibition and prostaglandin E2 release from syngeneic glomerular mesangial cells.

The concept of an active role of T lymphocytes in the initiation and development of autoimmune glomerulonephritis has gradually evolved from recent investigations. In the present study we started in a murine coculture system to directly examine cellular interactions of intrinsic glomerular mesangial cells (MC) and syngeneic T lymphocytes. Lymph node lymphocytes and, moreover, cloned T helper cells specifically affected syngeneic proliferating MC, causing growth inhibition and prostaglandin E2 (PGE2) release. The T cell specificity of mesangial cell responses was confirmed by demonstrating (i) that MC cocultured with other cell types showed no reaction and (ii) that additional activation of T lymphocytes by IL-2 or concanavalin A significantly enhanced the MC responses. Subsequently, we confirmed the presence of T cell factors in the supernatants responsible for the observed effects: interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF). Experiments with combinations of recombinant mouse IFN-gamma and human lymphotoxin or TNF-alpha showed that these lymphokines could substitute for the direct T lymphocyte effects causing a synergistic growth inhibition and PGE2 release from mouse MC. The observed lymphokine activities were not due to mesangiolysis as shown by neutral red uptake of MC. Pointing to the essential role of T helper cell-specific products, IFN-gamma antibodies abolished both the IFN-gamma and the combined IFN-gamma/TNF-alpha effect. Thus, our investigations with syngeneic MC-lymphocyte cocultures demonstrated that cultured MC specifically responded to T lymphocytes and their products.

Animals↗
PubMed1995

Thyroid cell survival in coculture with autologous peripheral or intrathyroidal lymphocytes.

OBJECTIVE: We have studied lymphocyte induced cytotoxicity and the production of interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) during coculture of thyrocytes and autologous lymphocytes from patients with Graves' disease and from normal subjects. PATIENTS: Thyroid tissues and lymphocytes were obtained from 28 patients with Graves' disease and from 9 control subjects. MEASUREMENTS: Lymphocyte induced cytotoxicity was evaluated on autologous thyrocytes using 5 metabolic tests: the MTT assay, the neutral red uptake, lactate dehydrogenase measurement and glutathione assay. IFN-gamma and TNF-alpha measurements were performed after 1, 5 or 7 days' coculture. RESULTS: The lymphocytes isolated from peripheral blood (PB lymphocytes) altered the morphology and the metabolism of autologous thyrocytes. The intrathyroidal lymphocytes isolated after Dispase digestion were not toxic whereas mechanically isolated lymphocytes exerted a little toxicity. No difference was seen between Graves' disease and normal cells. The supernatants from cocultures had higher IFN-gamma levels than those from lymphocyte cultures. In coculture, PB lymphocytes secreted more IFN-gamma and TNF-alpha than intrathyroidal lymphocytes. The PB lymphocyte induced cytotoxicity was not due to IFN-gamma and TNF-alpha alone. CONCLUSION: Peripheral blood lymphocytes are cytotoxic in vitro to autologous thyrocytes whereas intrathyroidal lymphocytes exert little or no cytotoxicity according to their isolation method. The mechanisms of lymphocyte induced toxicity remain to be explained.

Adult↗
PubMed1994

Determining periodontal ligament (PDL) cell vitality from exarticulated teeth stored in saline or milk using fluorescein diacetate.

One of the key factors for obtaining a favourable long-term prognosis in avulsed teeth is maintenance of the vitality of the periodontal ligament (PDL) cells. Most studies which have examined PDL cell vitality have used neutral red or trypan blue as stains. However, these stains have certain inherent disadvantages. The purpose of this paper was to (i) evaluate the use of saline and milk as storage media for PDL cells and (ii) determine the value of using fluorescein diacetate (FDA) as a staining medium for vital PDL cells on the root surface of avulsed teeth. Thirty-two single-rooted premolars were utilized from patients aged 13 to 28 years. Following atraumatic extraction, the teeth in the experimental groups were air dried for 10 min and then placed in either milk or saline for 120 min. Both control and experimental teeth were subjected to trypsinization procedures, staining with FDA, and haemocytometer readings to determine the number of vital cells. There was no statistically significant difference in the number of viable cells on the root surfaces of teeth after 2 h of storage in either milk or in saline. Furthermore, staining with FDA provided an excellent method by which to determine PDL cell vitality.

Adolescent↗
PubMed2006

Cytotoxicity of materials used in perforation repair tested using the V79 fibroblast cell line and the granulocyte-macrophage progenitor cells.

AIM: To compare the cytotoxicity of materials used to repair perforations using permanent V79 fibroblasts and murine granulocyte-macrophage progenitor cells (CFU-GM). METHODOLOGY: Set specimens from amalgam, glass-ionomer, SuperEBA, N-Rickert, MTA and gutta-percha were eluted with culture medium for 72 h and their cytotoxicities were assessed by incubating the extracts with V79 and bone marrow-derived progenitors for 24 h and 7 days, respectively. Cytotoxicity on V79 cells was judged using the total nucleic acid content (NAC), neutral red uptake (NRU) and reduction of the tetrazolium salt (MTT). The number of bone marrow CFU-GM colonies determined in clonal cultures stimulated with recombinant murine granulocyte-macrophage colony-stimulating factor was used to assess cytotoxicity to progenitor cells. Statistical analyses were conducted using the one-way analysis of variance and Tukey's test where appropriate. RESULTS: All materials were cytotoxic in both cell systems; however, CFU-GM was more sensitive to the extracts than V79 cells. A similar rank order of toxicity was observed in V79 cells using the NAC and the MTT assays: glass-ionomer > N-Rickert congruent with SuperEBA > gutta-percha > amalgam congruent with MTA (P < 0.05). In contrast, the NRU test exhibited a lower sensitivity to MTA, gutta-percha and amalgam extracts. In the clonal culture assay, the toxicity was less pronounced in the presence of gutta-percha, SuperEBA and MTA. Similar cellular responses were found by placing the set specimens directly in the clonal culture dishes. CONCLUSIONS: The sensitivity of toxicity depended on the choice of the endpoint and the cell-culture system. Nevertheless, MTA was ranked as the least cytotoxic cement in both cell systems.

Acrylic Resins↗
PubMed1989

Rapid impedance detection of salmonellas in confectionery using modified LICNR broth.

A commercially available broth, with the addition of inhibitors, was used for the rapid impediometric detection of salmonellas in confectionery. Pre-enrichment in skimmed milk was followed by lysine-iron-cystine-neutral red broth in a Bactometer 123 system. Results were obtained 3 d earlier than is possible with conventional microbiological tests. Some false positives were obtained predominantly with Citrobacter freundii, but this problem is also frequently encountered with traditional methods. Organisms responsible for false positives may be isolated and identified more rapidly than is possible by conventional methods.

Cacao↗
PubMed2006

FK506, a secondary metabolite produced by Streptomyces, presents a novel antiviral activity against Orthopoxvirus infection in cell culture.

AIMS: To investigate the antiviral potential of the macrolide FK506, produced by Streptomyces tsukubaensis, against Orthopoxvirus infection in cell culture, and determine the replicative stage of viral cycle affected by the treatment. METHODS AND RESULTS: Cell lines were infected with different Orthopoxviruses and treated with FK506. The macrolide inhibited the replication of the prototypic Orthopoxvirus, vaccinia virus strain WR, with an IC50 of 12.05 micromol l(-1). Progeny production of other Orthopoxviruses was also inhibited by FK506 at noncytotoxic concentrations, as evaluated by the neutral-red uptake assay and metabolic labelling of cellular proteins. By Western blot assay, we detected a severe inhibition (approximately 87.6% +/- 2.78%) of VV strain WR post-replicative protein synthesis. A similar reduction of virus DNA accumulation, as observed by slot-blot assay, probably accounts for the subsequent inhibition of virus late proteins. CONCLUSIONS: The macrolide FK506, isolated from S. tsukubaensis, presents a novel anti-poxvirus activity, probably targeting the stage of DNA replication during Orthopoxvirus infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The secondary metabolite FK506, isolated from the culture filtrate of S. tsukubaensis, shows a pleiotropic range of activities, and might be a valuable tool as a lead structure in the generation of non-immunosuppressant analogues with strong anti-poxvirus activity.

Animals↗
PubMed1989

An investigation of the fixation and staining of lipids by a combination of malachite green or other triphenylmethane dyes with glutaraldehyde.

Mixtures of the monocationic triphenylmethane dyes, malachite green or crystal violet, with glutaraldehyde, retained and stained phospholipid droplets in chloroplasts of leaves of Lolium multiflorum Lam. These dyes also stained trilinolenin; phosphatidic acid dioleoyl, dipalmitoyl; phosphatidylcholine dioleoyl, dilinoleoyl; and phosphatidylethanolamine dioleoyl on filter paper models. In this model system the dipalmitoyl derivatives of phosphatidylcholine and phosphatidylethanolamine did not stain well, if at all. Washing of the dyed samples with 0.5 M sodium chloride solution did not remove the colour, suggesting that the interaction is unlikely to be purely ionic. Except with trilinolenin, the colour and possibly the lipid samples were removed from the filter paper model system on washing with 100% ethyl alcohol. Other triphenylmethane dyes (methyl green, light green and fast green FCF) did not retain phospholipid droplets in tissue. Fast green did, however, stain phospholipids in the model system. Two quinone-imine dyes, neutral red and toluidine blue O, while staining phospholipids in the model system did not retain droplets on the chloroplasts but did assist in the retention and staining of cell membranes. The basis of the reaction between lipid and dye is discussed in relation to the structural formulae of the dyes and model lipids. It is possible that there is an interaction between the hydrophobic fatty acid ester side chains of the lipid and the dyes. Neither the phosphate nor the polyhydric alcohol moieties of the lipid seem to be essential for staining or retention of lipid.

Fixatives↗
PubMed1995

Ovariolar 'basal body' development and physiological age of the mosquito Aedes aegypti.

Dissection of the ovaries of the mosquito Aedes aegypti (Diptera: Culicidae) revealed, in each ovariole, a group of seven to nine specialized epithelial cells in a region of the calyx wall that is enclosed by the end of the ovariolar sheath. This group of cells is termed the basal body. During ovulation, the mature oocyte passes from the ovariole into the calyx lumen through the basal body. Subsequently, granulation occurs in the basal body cells. The granular basal bodies differ from all previously described ovarian structures. In multiparous females the size and optical density of the granular basal bodies increase after each ovulation. Examination of the granular basal bodies in intact ovaries, stained with neutral red, provides an easy method for distinguishing parous from nulliparous females, and has potential as a new method of age grading.

Aedes↗
PubMed2005

Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.

Basal resistance in plants is induced by flagellin and several other common bacterial molecules and is implicated in the immunity of plants to most bacteria and other microbes. However, basal resistance can be suppressed by effector proteins that are injected by the type III secretion system (TTSS) of pathogens such as Pseudomonas syringae. This study demonstrates that basal resistance in the leaves of Nicotiana benthamiana is accompanied by reduced vascular flow into minor veins. Reduced vascular flow was assayed by feeding leaves, via freshly excised petioles, with 1% (weight in volume, w/v) neutral red (NR) and then observing differential staining of minor veins or altered levels of extractable dye in excised leaf samples. The reduced vascular staining was localized to tissues expressing basal resistance and was observable when resistance was induced by either the non-pathogen Pseudomonas fluorescens, a TTSS-deficient mutant of P. syringae pv. tabaci, or flg22 (a flagellin-derived peptide elicitor of basal resistance). Nicotiana benthamiana leaf areas expressing basal resistance no longer elicited the hypersensitive response when challenge inoculated with P. syringae pv. tomato DC3000. The reduced vascular staining effect was suppressed by wild-type P. syringae pv. tabaci and P. fluorescens heterologously expressing a P. syringae TTSS and AvrPto1(PtoJL1065). TTSS-proficient P. fluorescens was used to test the ability of several P. syringae pv. tomato DC3000 effectors for their ability to suppress the basal resistance-associated reduced vascular staining effect. AvrE(PtoDC3000), HopM1(PtoDC3000) (formerly known as HopPtoM), HopF2(PtoDC3000) (HopPtoF) and HopG1(PtoDC3000) (HopPtoG) suppressed basal resistance by this test, whereas HopC1(PtoDC3000) (HopPtoC) did not. In summary, basal resistance locally alters vascular function and the vascular dye uptake assay should be a useful tool for characterizing effectors that suppress basal resistance.

Bacterial Proteins↗
PubMed1991

Spatial variation of sequestered calcium in the multicellular stage of Dictyostelium discoideum as assayed by chlortetracycline fluorescence.

We have used chlortetracycline (CTC) as a fluorescent probe to detect the distribution of sequestered calcium in multicellular stages of Dictyostelium discoideum. Tips of late aggregates, slugs and early culminating masses fluoresce very strongly. Most of the fluorescence is intracellular in origin and emanates from a small number of intense punctate sources. The sources correspond in part to autophagic vacuoles vis. neutral-red staining, acidic digestive vesicles, and may also include intracellular organelles; cytoplasmic fluorescence is much weaker in comparison. The level of fluorescence drops in the middle portion of slugs and rises again in the posteriormost region, though not to as high a level as in the tip. This holds good irrespective of whether CTC is applied only in the neighbourhood of the aggregate centre, only in the aggregate periphery, or to the whole aggregate. We infer that there must be a good deal of mixing in the stages leading from aggregation to slug formation; thus the serial order in which cells enter an aggregate does not bear any relation to their ultimate fates. The other implication of our study is that calcium sequestration is much more extensive in prestalk and anterior-like cells than in prespore cells. These findings are discussed with regard to possible implications for pattern formation.

Animals↗
PubMed2004

Itraconazole corneofungimetry bioassay on Malassezia species.

Yeasts of the genus Malassezia are part of the normal skin biocenosis and are involved in a series of distinct skin disorders and specific dermatomycoses in man and animals. Several species are currently distinguished. Their relative in vitro susceptibility to antifungals appears different according to the species and to the nature and route of administration of the drug. Corneofungimetry is an ex vivo bioassay allowing to test the fungal response on human stratum corneum following oral intake of a given antifungal by volunteers. Two series of cyanoacrylate skin surface strippings (CSSS) were harvested from the volar forearm of 30 volunteers before and after a 2-week treatment with itraconazole 200 mg daily. They were coated by olive oil and inoculated with suspensions of seven different Malassezia spp. After a 1-week culture on CSSS, the amount of viable yeasts was assessed using neutral red staining assisted by computerized image analysis. Growth of the seven species was not similar on the CSSS from untreated stratum corneum. The ranking order from the most proliferative to the least was M. restricta, M. sympodialis, M. globosa, M. furfur, M. obtusa, M. slooffiae and M. pachydermatis. Their growth was abated to almost the same level after itraconazole treatment. It is concluded that in vivo treatment with itraconazole is highly active against all Malassezia spp. colonizing the human stratum corneum.

Administration, Oral↗