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[Tenotoxic potential of fluoroquinolones in the choice of surgical antibiotic prophylaxis in ophthalmology].

PURPOSE: Fluoroquinolones are mainly used in ophthalmic antibiotic prophylaxis because of their broad spectrum activity and good ocular diffusion. But a single oral dose of fluoroquinolones can result in a serious source of tendinopathy and tendon rupture, especially in patients 60 years and older. It seems very important to investigate tendon toxicity of fluoroquinolones to improve the risk-benefit ratio in ophthalmologic antibiotic prophylaxis. MATERIAL: and methods: The intrinsic tenotoxic potential of four fluoroquinolones (pefloxacin, ofloxacin, ciprofloxacin, levofloxacin) was directly evaluated on living adherent tendon cells in microplates. Cell viability and reactive oxygen species production was evaluated using neutral red, alamar blue, and dichlorofluorescin diacetate tests. RESULTS: Results showed a loss of viability associated with free radical production depending on fluoroquinolone molecules. Pefloxacin appeared more tenotoxic but no study has confirmed its efficacy in surgical antibiotic prophylaxis and its use in the patient who is 60 years and older could be disputed. Ciprofloxacin is highly toxic with a low ocular diffusion and seems to be inappropriate for antibiotic prophylaxis. Ofloxacin and levofloxacin are less cytotoxic, associated with good ocular diffusion and a broad antibacterial spectrum. CONCLUSION: Ofloxacin and levofloxacin seem to be good alternatives for improving the risk-benefit ratio in surgical antibiotic prophylaxis in patients 60 years and older.

Achilles Tendon↗

[Promoter effect of mercury chloride and methyl-mercury on human keratinocytes in culture].

Mercury has received considerable media focus because it is present in dental amalgams and seafood. There is potential exposure in gas meters, thermometers and fluorescent lamps workers. To evaluate its possible epigenetic carcinogen effect, cultures of human keratinocytes were treated with increasing doses of HgCl2 for 30 min, 24 h and of CH3HgCl for 24 h, respectively. The red neutral method was used to evaluate the doses of HgCl2 and CH3HgCl which had no cytotoxic effect. Then, the dye transfer method was used to investigate the gap junctions-mediated intercellular communication (GJIC). Cells were microinjected with Lucifer Yellow CH by using the Eppendorf Apparatus and the Leica inverted microscope. After 30 min incubation at the concentration of 10 microM, HgCl2 did not exert inhibition of GJIC. Conversely, after 24 h at the concentration of 10 nM, HgCl2 inhibited GJIC. Incubation with CH3HgCl at the concentration of 250 nM for 24 h reduced the number of fluorescent cells, thus denoting a inhibition of GJIC. Taken together our data demonstrated that: i) HgCl2 and CH3HgCl exerted an inhibitory effect upon GJIC; ii) HgCl2 resulted to inhibit GJIC at concentrations 25 folds lower than CH3HgCl. Further studies will be addressed to evaluate whether the reversal of GJIC inhibition could be obtained by withdrawal of toxic substance, or by the addition of a GJIC activator like the retinoic acid. Finally to shed light on the possible effect of mercury derivates at the transcriptional or translational levels, the expression profile of the connexin 43 gene after HgCl2 and CH3HgCl exposure of cultured human keratinocytes will be investigated.

Cell Communication↗

[Effects of testosterone on proliferation and IgG production of splenic lymphocytes in vitro].

The purpose of the present study was to investigate the effects of testosterone on proliferative response and IgG production by splenic lymphocytes in vitro. Splenic lymphocytes from rats were cultured in the presence of the indicated doses of testosterone propionate for 72 h. The cell viability was assessed by a trypan blue exclusion test. Proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and supravital stain neutral red assay. Levels of IgG and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). The proliferative response and IgG production of splenic lymphocytes in the presence of testosterone were lower than those of control cells. Testosterone may inhibit proliferative response and IgG production of splenic lymphocytes. The results also support that androgen may play an important role in immune regulations.

Animals↗

Optical imaging of long-term depression in the mouse cerebellar cortex in vivo.

Conjunctive stimulation of climbing fiber and parallel fiber inputs results in long-term depression (LTD) at parallel fiber-Purkinje cell synapses. Although hypothesized to play a major role in cerebellar motor learning, there has been no characterization of the cellular and molecular mechanisms of LTD in the whole animal, let alone its spatial properties, both of which are critical to understanding the role of LTD in cerebellar function. Neutral red optical imaging of the cerebellar cortex in the anesthetized mouse was used to visualize the spatial patterns of activation. Stimulation of the parallel fibers evoked a transverse beam of optical activity, and stimulation of the contralateral inferior olive evoked parasagittal bands. Conjunctive stimulation of parallel fibers and climbing fibers induced a long-term decrease (at least 1 hr) in the optical response to subsequent parallel fiber activation confined to the region of interaction between these two inputs. Activation of climbing fibers alone failed to induce the long-term decrease. Field potential recordings confirmed that the depression is postsynaptic and restricted to the interaction site. The long-term depression in the beam was prevented by a group 1 metabotropic glutamate receptor (mGluR(1)) antagonist and was absent in transgenic mice selectively expressing an inhibitor of protein kinase C (PKC) in Purkinje cells. Conversely, the long-term depression occurred in the mGluR(4) knock-out mouse, consistent with its postsynaptic origin. In addition to providing the first visualization of parallel fiber-Purkinje cell LTD in the cerebellar cortex, this study demonstrates the spatial specificity of LTD and its dependence on mGluR(1) and PKC in vivo.

Animals↗

Cellular viability and ultrastructure of stented, antibiotic sterilized and cryopreserved sheep biological valves implanted for one year in tricuspid position.

UNLABELLED: The aim of the study was an evaluation of viability and damage of biological cells sterilized with antibiotics and cryopreserved a year after the implantation. Sheep antibiotic sterilised and cryopreserved biological valves were implanted in tricuspid position in young sheeps for one-year period. After this time the valves removed and studied morphologically. The control group consisted of 7 intact valves, the comparative group, so called group of valves after the processing antibiotic sterilization and cryopreservation consisted of 7 valves after mentioned procedures. Histologic investigations were based on paraffin sections of formalin-fixed valve cusps, stained with H&E and Masson trichrome. Valve viability was assessed using intravital staining with fluoresceine diacetate, whereas damaged cells were visualized by intravital staining with neutral red. Additionally, the ultrastructural studies were performed. The viability and ultrastructural results were compared with the pathologic process in the valve. CONCLUSIONS: 1. preliminary preparation with antibiotic sterilisation and cryopresrevation induces valve leaflet oedema and degenerative ultrastructural processes, colliquative cell necrosis and apoptotic morphology of a part of valve cells; 2. cryopreserved and antibiotic sterilised pulmonary valve after one-year implantation behaves lining cells, but ultrastructural changes indicates many degenerative phenomena in smaller degree than after preparation, sterilization and cryopreservation. Histopathologically the degenerative changes were prevalent.

Animals↗

The Bactericidal And Cytotoxic Effects Of Antimicrobial Wound Cleansers.

OBJECTIVE: Wound care is a part of daily activity for many athletic trainers. Knowing which cleansers are effective against the bacteria that most commonly cause infection and whether they are toxic to healthy cells enables athletic trainers to make educated decisions on which cleanser to use. We compared the bactericidal effectiveness and cytotoxicity to human fibroblast cells of 4 cleansers at various dilutions. DESIGN AND SETTING: A 4 x 4 factorial design was used for the cytotoxicity testing. The independent variables were type and dilution of cleanser. The dependent variable was cell viability of the human fibroblast cells. We used a 2 x 3 x 4 x 4 factorial design for the bacterial testing. The independent variables were type and dilution of bacteria and type and dilution of cleanser. The dependent variable was the bactericidal action of the cleanser on the bacteria. SUBJECTS: Human foreskin samples were used to obtain a line of fibroblast cells. Bacterial samples were obtained from an athletic training clinic, isolated from swabs of a whirlpool water supply valve (Pseudomonas aeruginosa) or skin surface (Staphylococcus aureus). MEASUREMENTS: We obtained bactericidal measurements by testing isolated Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. Minimum and maximum concentrations were identified according to bactericidal effectiveness. Cytotoxicity measurements were obtained from spectrophotometer readings of a neutral red assay for fibroblast cell viability. Final dilutions tested were determined by pilot testing. RESULTS: At the 1:5 dilution of product in sterile 0.9% saline, both Cinder Suds and Nitrotan and hydrogen peroxide were different from the control with regard to Pseudomonas aeruginosa. At the 1:10 dilution, both Betadine and hydrogen peroxide were different from the control with regard to Pseudomonas aeruginosa. These 2 cleansers were also different from each other. At the 1:10 dilution, only Betadine was not different from the control for the cytotoxicity testing. CONCLUSIONS: Betadine was both effective against bacteria and not harmful to human fibroblast cells at a 1:10 dilution of a commercially purchased solution.

Journal Article↗

Spermiophages in human semen in normal subjects and in patients with selected diseases.

In order to screen for spermiophages in human semen, a Neutral Red Test was carried out in: a) normal subjects (NORMAL); b) male partners of infertile couples (INFERT); c) subjects with selected diseases. Spermiophages were observed only in 7.5% of the normospermic subjects, while the test was positive in 26.5% of INFERT. Positivity was higher in the selected pathological groups, even compared to the INFERT group. This observed increase in spermiophage activity could be an important pathogenetic factor in some cases of male infertility.

Adult↗

CRESYL BLUE AND CRESYL BLUE PLUS SUDAN 3 AS STAINS FOR USE IN EXFOLIATIVE AND OTHER CYTOLOGY.

Investigating the value of a number of supravital strains for use in exfoliative cytology, the authors found cresyl blue (1% solution in distilled water), with and without Sudan III (0.3% in 70% alcohol), to be reliable and relatively long-lasting. Specimens stained with cresyl blue and Sudan III can be kept for one or two days, or for longer periods (up to 3 weeks or more) if a moist chamber is used containing filter-paper moistened with 10% formol to prevent fungal contamination. These stains are less toxic than Janus green and simpler to use than neutral red. The minimum of simple laboratory apparatus is needed.During 1960-62, 1955 specimens from 460 hospitalized patients in Beer-Yaacov, Israel, were stained with these two preparations. Cytological examination led to a diagnosis of cancer in 16% of the patients, with only 9 false diagnoses.

Azo Compounds↗

[Macrophages in human semen].

OBJECTIVES: To investigate the presence of macrophages in human semen samples and the function they carry out in the seminal fluid. Their presence was studied in relation to spermatic morphology, percentage of spermatozoids with native DNA, and presence of antispermatic antibodies. METHODS AND RESULTS: The work was performed with semen samples from 31 unfertile males from 63 couples in which the "female factor" was ruled out as the cause of infertility. Sperm study according to WHO (1992) was carried out in all samples, in addition to: DNA study with acridine orange as fluorocrom, macrophage concentration by neutral red in a Neubauer camera, and detection of antispermatic antibodies with a mixed agglutination test (TAC II) (validated with Mar Screen-Fertility technologies). Sperm morphology was evaluated by Papanicolaou test. 19/31 selected sperm samples (61.3%) showed increased concentration of macrophages, 13 of them (41.9%) with denaturalized DNA, and 8 (25.8%) abnormal morphology. Six samples showed increased macrophage concentration and predominance of native DNA, whereas 11 samples showed increased macrophages and abnormal morphology. Among 18 (58.1%) samples showing antispermatic antibodies 14 (77.7%) had an increased concentration of macrophages. Statistical analysis resulted in a high correlation between macrophage concentration and increased percentage of spermatozoids with denaturalized DNA (p < 0.05). An increased concentration of macrophages is associated with the presence of antispermatic antibodies (p < 0.05). There was not evidence of significant association between concentration of macrophages and percentage of morphologically normal spermatozoids (p < 0.05). CONCLUSIONS: We can conclude that macrophages are present in human semen and participate in immunovigilance contributing to improve the seminal quality.

Cell Count↗

[Application of terrestrial invertebrates biomarkers in soil pollution ecology study].

Biomarkers in terrestrial invertebrates play an important role in estimating either exposure or resultant effects of pollutants in soil ecosystem, which has received increasing attention and made significant progress. The present paper has drawn three important biomarkers, i.e., lysosomes, stress protein and mettllothioneins (MTs). The lysosomal membrane stability of coelomocytes was assayed as neutral-red retention time (NRR-time) resulting from toxicant stress. Hsp70 and Hsp60 were commonly used in the stress protein families. The quantification methods of different isofoms of the mettllothioneins could specifically indicate different metal contamination. The rationale, characteristics, and relevant exemplary case of assaying the biomarkers and their promising application to ecotoxicological diagnosing in soil contamination were discussed.

Animals↗

Antitumor and anti-angiogenic activity of Ganoderma lucidum polysaccharides peptide.

AIM: To investigate the antitumor and anti-angiogenic activity of Ganoderma lucidum polysaccharides peptide (GLPP). METHODS: Antitumor effect of GLPP was observed in tumor-bearing mice in vivo. At the same time, the effects of GLPP on proliferation of tumor cells and human umbilical cord vascular endothelial cell (HUVEC) were detected by MTT assay in vitro. Subsequently, spleen lymphocytes proliferation of nude mice was stimulated by LPS or ConA. To investigate the anti-angiogenic effect of GLPP, GLPP 80 microg per disc and GLPP-treated serum 10 microL per disc were added to the chick chorioallantoic membrane (CAM) respectively in vivo. RESULTS: GLPP 50, 100, and 200 mg/kg inhibited growth of Sarcoma 180 in BALB/c mice markedly by 35.2 %, 45.2 %, and 61.9 %, respectively. GLPP which was directly added to the cultured medium did not inhibit PG cell proliferation in vitro; but GLPP-treated serum 50, 100, 200 mg/kg potently inhibited PG cell proliferation by 22.5 %, 26.8 %, and 30.3 %, respectively; and reduced the xenograft (human lung carcinoma cell PG) in BALB/c nude mice greatly in vivo by 55.5 %, 46.0 %, and 46.8 %, respectively. Lymphocytes proliferation of nude mice could be stimulated by LPS 5 mg/L but not by ConA 2.5 mg/L, indicating that GLPP could not promote the T lymphocyte proliferation and neutral red phagocytosis of peritoneal macrophages of nude mice. The CAM assay showed that GLPP and GLPP-treated serum had anti-angiogenic effect. GLPP (1, 10, and 100 mg/L) inhibited HUVEC proliferation in vitro with the inhibitory rate of 9.4 %, 15.6 %, and 40.4 %, respectively. CONCLUSION: GLPP has antitumor and anti-angiogenic activity. The anti-angiogenesis of GLPP may be a new mechanism underlying its anti-tumor effects.

Angiogenesis Inhibitors↗

[Activation of the immunologic function of rat Kupffer cells by the polysaccharides of Angelica sinensis].

AIM: To observe the influence of polysaccharides of Angelica sinensis (ASP) on the immunologic function of rat Kupffer cells. METHODS: Normal rat Kupffer cells were treated with ASP in vitro. Absorbance at 540 nm ( A540) of neutral red absorption and supernatant NO, TNF-alpha in the cells were measured to evaluate the immunologic function of Kupffer cells; LDH leakage was measured to estimate the severity of cellular damage; Rats were given ASP 0.025, 0.1, 0.25 and 1.0 g x kg(-1) ig (qd x 7 d) in vivo. The above indices and ACP of Kupffer cells were measured, sGST and sALT activity were detected as indices of hepatotoxicity. RESULTS: ASP markedly enhanced the phagocytic activity, ACP and supernatant NO, TNF-alpha of Kupffer cells both in vitro and in vivo . The increase of sGST was observed after administration of ASP 1.0 g x kg(-1), but the LDH leakage of the hepatocytes was not increased in vitro. CONCLUSION: ASP with suitable dose could activate the function of Kupffer cells. Slight liver injury was caused by ASP 1.0 g x kg(-1) in vivo, which was likely caused by factors, such as NO, TNF-alpha, indirectly.

Adjuvants, Immunologic↗

[Modified sperm stress test. Immune response and functionality of the human spermatozoid membrane].

OBJECTIVE: Our purpose was to study, in semen samples of infertile patients, the relationship between the Modified Sperm Stress Test (MOST) and the presence of antisperm antibodies (ASA), macrophages concentration and the Hypoosmotic Swelling Test (HOST). METHODS: Semen samples from 42 men undergoing evaluation for infertility were examined according to WHO criteria. Twenty-five of them, whithout clumping non hyperviscosity, were selected. The MOST test was applied according to the author's original technique. ASA were determined with a direct mixed agglutination test, TAC II, developed and validate by our group. Macrophage concentration was evaluated with Neutral Red stain, and functional integrity of the sperm membrane with the Hypoosmotic Swelling Test using an hypoosmotic solution of 150 mOm/ml composed of equal parts of fructose and sodium citrate. RESULTS: The Chi square test was applied to the observational data obtaining the following results: There was a statistically significant association between the presence of ASA and altered MOST (p<0.001). In all samples with ASA, abnormal MOST values were obtained (MOST<0.39). Besides, there is a statistically significant association exists between the increased concentration of macrophages and abnormal MOST (p<0.01); and altered HOST was positively correlated with abnormal MOST (p<0.02). CONCLUSION: Results clearly demonstrate the high predictive power of MOST like a test of sperm resistance to the forced lipoperoxidation, offering conditions to become a good predictor of sperm performance. Understanding the sperm resistance to the ROS and their harmfull effects on the sperm functions, a proportion of infertile men can be succesfully treated.

Autoantibodies↗

In vitro cytotoxicity evaluation of biomaterials on human osteoblast cells CRL-1543; hydroxyapatite, natural coral and polyhydroxybutarate.

The aim of this study was to evaluate the in vitro cytotoxicity of biomaterials; Hydroxyapatite (HA), Natural coral (NC) and Polyhydroxybutarate (PHB). Three different materials used in this study; HA (Ca10(PO4)6(OH)2), NC (CaCO3) and PHB (Polymer) were locally produced by the groups of researcher from Universiti Sains Malaysia. The materials were separately extracted in the complete culture medium (100mg/ml) for 72h and introduced to the osteoblast cells CRL-1543. The viability of osteoblast CRL-1543 cultivated with these extraction materials after 72h incubation period was compared to negative control with neutral red assay by using spectrophotometer at 540nm. The results showed the non-cytotoxicity of the materials. After 72h of incubation period, HA showed 123% viable cells, NC was 99.43% and PHB was 176.75%. In this study, cytotoxicity test dealt mainly with the substances that leached out from the biomaterial. The results obtained showed that the materials were not toxic and also promoted cells growth in the sense of biofunctionality.

Animals↗

Comparative in vitro studies of the potentiation of tumor necrosis factor (TNF)-alpha, TNF-beta, and TNF-SAM2 cytotoxicity by hyperthermia.

Hyperthermia can strikingly enhance tumor necrosis factor-alpha (TNF-alpha) cytotoxicity in vitro and in vivo. Other forms of TNF may have tumor therapeutic applications and their interaction with hyperthermia should also be assessed. We have compared the effect of heat on the in vitro cytotoxic response of murine L929 and EMT-6 and human T24 tumor cells to three TNF forms; recombinant human TNF-alpha, TNF-beta (lymphotoxin), and TNF-SAM2. A neutral red assay was used to measure toxicity at 18-20 h after initiating the heat treatment. TNF treatment preceded heating by 0-4 h or followed it by 2 h. Heating was done at 39 or 40.5 degrees C for 24 h, 40.5 or 42 degrees C for 1 h, or 43 degrees C for 1-1.5 h. We found that both TNF-beta and TNF-SAM2 toxicities, like that of TNF-alpha, were markedly enhanced by hyperthermia. Neither EMT-6 nor T24 cells responded consistently to any of these TNFs at heat doses up to 1 h at 43 degrees C, but an increment of only 15 min more at 43 degrees C sensitized EMT-6 cells and 1.5 h at 43 degrees C resulted in extensive EMT-6 cell killing. The T24 cells remained resistant except for variable responses at the highest TNF and heat doses. If TNF treatment was begun immediately before or 2 h after beginning to heat the EMT-6 cells, sensitization was reduced or eliminated, respectively, for all three TNF forms relative to protocols in which TNF was added 1, 2, or 4 h before heating.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Effects of exopolysaccharide from Tolypocladium sinense on murine immunocytes in vitro].

OBJECTIVE: To elucidate the effects of exopolysaccharide from Tolypocladium sinense on murine immunocytes in vitro. METHODS: Extracted exopolysccharide (EPS) from cultured Tolypocladium sinense. The effects of EPS on mouse immune parameters were detected in vitro using cell culture techniques. RESULTS: The results indicated that EPS might significantly not only promote mouse macrophage neutral red uptake ability and cytotoxicity (P < 0.05), but also stimulate their thymus lymphocytes proliferation (P < 0.05). CONCLUSION: EPS may elevate murine cellular immunity to a certain.

Animals↗

Rapid chromatographic methods for the identification and determination of chlorophenoxy herbicides.

Derivatives of chlorophenoxy carboxylic acids have been the first class of herbicides in continuous use since 1947. The main interest for these substances is due to their evident chronic toxicity and carcinogenic effect. On the other hand, they can cause acute toxicity and have significant role in suicidal attempts. In this paper we have investigated analytical approaches that could be used for rapid identification and determination of chlorophenoxy herbicides in modestly equipped laboratories for the clinical toxicology. Thin layer chromatography on cellulose layer using neutral red as ion pairing reagent gave the best results in separation of different herbicides, making possible visualisation at the daylight without further reagents or equipment. High performance liquid chromatography (HPLC) methods for separation on C8 and C18 phases with and without ion-pairing reagent were compared. It was found that HPLC on C18 phase utilising ion-suppression mode has the best reproducibility, linearity and mass limit of detection suitable for quantification of chlorophenoxy herbicides after acute poisoning.

2,4-Dichlorophenoxyacetic Acid↗

Cytochemical properties of Botryllus schlosseri haemocytes: indications for morpho-functional characterisation.

In the present study, we carried out a detailed light microscopy investigation of the cytochemical properties of the haemocytes of the colonial ascidian Botryllus schlosseri, using new cytochemical stains and enzymatic markers, a panel of antibodies and lectins as probes to characterise Botryllus blood cells further. Results indicate that lymphocyte-like cells are circulating undifferentiated cells recognised by anti-CD34 antibody and there are at least two defined haemocyte differentiation pathways: i) phagocytes, represented by hyaline amoebocytes and macrophage-like cells, which share similar staining properties, the same hydrolytic enzyme content as well as the presence of detectable cytochrome-c-oxidase activity, recognition by anti-CD39 and Narcissus pseudonarcissus agglutinin; ii) cytotoxic cell line, represented by granular amoebocytes and morula cells which have vacuoles stained by Ehrlich's stain and Neutral Red; DOPA-containing protein are present inside morula cell vacuoles. Pigment cells and nephrocytes are involved in catabolite storage but their relationships with other cell types are less clear.

Animals↗