In vitro sensitivity of human gastric cancer cells (HGC-27) to Helicobacter pylori cytotoxin.

BACKGROUND: The VacA cytotoxin produced by Helicobacter pylori is considered an important co-factor in the pathogenesis of chronic gastritis, peptic ulcer and gastric carcinoma. The toxin remains partly bound on the bacterial surface, but a certain amount is secreted and can bind receptors on gastric epithelium. The vacuolizing activity of this toxin is related to alteration of endo-lysosomic function and pore formation into plasmatic membrane. METHODS: We investigated the 'in vitro' effect of filtrates obtained from two broth cultures of H. pylori with different genotype (vacA+ and vacA-) as verified by PCR. The effect was studied on three cell lines of epithelial origin: HeLa cells (reference strain for testing vacuolization), human transformed keratinocytes HaCaT, human gastric carcinoma cells HGC-27, and on a murine leukaemia WEHI-3B. The filtrate concentrations capable of giving vacuolization (NRU test), antiproliferative and cytotoxic effects (MTT test) were determined. The modulating effect of filtrates on drug toxicity was investigated on HeLa and HGC-27 cells by testing topoisomerase inhibitors (Ciprofloxacin and Camptothecin) and non-steroidal anti-inflammatory molecules (Aspirin and Indomethacin). RESULTS: Our results confirm that vacuolizing activity is present only in VacA+ filtrate and that HaCaT and HeLa cells show a similar sensitivity, whereas gastric HGC-27 cells appear significantly resistant to VacA+ activity. Although VacA filtrate does not produce vacuolisation, it affects the cell proliferation and is cytotoxic to the four cell lines. Both the VacA+ and VacA- filtrates (at non-cytotoxic concentrations) produce a decrease in drug toxicity with the unique exception of Ciprofloxacin to gastric HGC-27 cells, which in the presence of VacA+ and VacA- produces a significant increase in toxicity. CONCLUSIONS: These data suggest that products from H. pylori (other than those that have antiproliferative and toxic activity) may modulate the sensitivity of cells to drugs 'in vitro'. If this also occurs 'in vivo', we can assume that H. pylori products interfere with drug activity on gastric tissue and also with other factors (such as cytokines) with a role in the genesis of diseases in which Helicobacters are potentially involved.

High sensitivity of human epidermal keratinocytes (HaCaT) to topoisomerase inhibitors.

In the panorama of the numerous established cell lines, the human keratinocyte line HaCaT has a very interesting feature, having a close similarity in functional competence to normal keratinocytes. This cell line has been used in many studies as a paradigm for epidermal cells and therefore we selected HaCaT as a cell model for investigating the activity of three antitopoisomerase drugs (Camptothecin, Doxorubicin, Ciprofloxacin) on in vitro cell growth. The effect was evaluated both by a 24-h cytotoxicity test and by a 7-day antiproliferation assay, in which the cell viability was assessed by an MTT (3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenil-2-H-tetrazolium bromide) test. DNA topoisomerase I was also partially purified from a nuclear extract of HaCaT cells, the level of topo I catalytic activity was measured by a pBR322 DNA relaxation assay and then the in vitro effect of antitopoisomerase drugs on the target enzyme was also assessed. The results indicated that the in vitro sensitivity of human epidermal HaCaT cells to antitopoisomerase drugs is comparable to that of many human tumour cell lines. HaCaT cells express a high level of topoisomerase I activity that is significantly inhibited by both Camptothecin and Doxorubicin and to a minor degree by Ciprofloxacin. A high correlation between the cell sensitivity to the antitopoisomerase I drug measured by the MTT test and the in vitro direct inhibition of HaCaT topoisomerase I was observed, suggesting that HaCaT cells can represent a very interesting model both for studying cellular pharmacokinetics of antineoplastic drugs on keratinocytes and for predicting possible secondary effects, exerted by these drugs on cutaneous cells, during treatment with chemotherapy.

Altered DNA-cleavage activity of topoisomerase II from WEHI-3B leukemia cells with specific resistance to ciprofloxacin.

In order to investigate the mechanisms of drug resistance arising in tumor cells, we investigated the capacity of fluoroquinolones to inhibit the in vitro growth of WEHI-3B monomyelocytic leukemia cells and then we established a variant of this line (currently maintained in the absence of drug). The line, named WEHI-3B/CPX, expresses a specific resistance to ciprofloxacin (CPX; resistance index=17.3+/-2.2), and does not show cross-resistance with other fluoroquinolones, camptothecin and topoisomerase II inhibitors such as doxorubicin, etoposide and teniposide. Although a little decrease in intracellular accumulation of CPX is observed in WEHI-3B/CPX cells, these cells do not express MDR or LRP markers, and the resistance is not circumvented by verapamil. Purified nuclear extracts from WEHI-3B and WEHI-3B/CPX cells were tested for topoisomerase I catalytic activity and checking in vitro topoisomerase I sensitivity to CPX and camptothecin inhibition, but no difference was observed. As the treatment with CPX showed that the resistant cell line suffers a significantly lower number of breaks in the DNA molecule we also addressed our investigations to the topoisomerase II-dependent DNA cleavage that, in the resistant clone, was found dramatically less susceptible to be enhanced by CPX both in pre-strand and post-strand DNA passage conditions. WEHI-3B/CPX cells do not express any character of multidrug resistance and represent a rare case of specific drug resistance to CPX. The specific resistance to CPX observed in these cells is related to a functional decrease of topoisomerase II cleavage activity. It could be consequent to a decreased binding affinity of CPX for the topoisomerase II--DNA complex or to a decreased affinity or specificity of topoisomerase II for its DNA cleavage sites.

Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria.

BACKGROUND: Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state. A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment. METHODS: We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis. After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex. A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome. Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively. RESULTS AND CONCLUSIONS: With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology.

Role of SR-4987 stromal cells in the modulation of doxorubicin toxicity to in vitro granulocyte-macrophage progenitors (CFU-GM).

Bone marrow stromal microenvironment is essential for the maintenance of the hematopoietic stem cell renewal both by cell-cell interaction and cytokine production. However, stromal cells also exhibit drug metabolizing activities and they may accumulate the drug and successively affect hematopoietic progenitors by a retarded release. Our study investigated the role of both primary culture of murine bone marrow stroma and established stromal cells (SR-4987) in modulating the "in vitro" toxic activity of Doxorubicin (DXR) against murine granulocyte-macrophage progenitors (CFU-GM). The main part of the study has been performed by a "in vitro" agar bilayer technique based on the CFU-GM assay performed over a feederlayer of stromal cells. The results suggest that bone marrow stromal cells play also an important role in decreasing the toxicity of Doxorubicin. Further SR-4987 stromal cells produce a Doxorubicin metabolite (not belonging to the series of metabolites described in literature) which is completely ineffective in inhibiting the growth of CFU-GM and the activity of topoisomerase I. Our data suggest that bone marrow stromal cells must be considered as a cell population having opposite pharmacological roles in modulating the drug toxicity on hematopoietic progenitors. In our model a mechanism of detoxification concerns the capacity of SR-4987 stromal cells to inactivate the drug. For a better prediction of drug hematotoxicity, it is very important to develop "in vitro" cell models able to discriminate between positive and negative modulation of drug toxicity that stromal cells can exert in the bone marrow microenvironment.

SR4987 and L1210 cell lines: two models in which cholera toxin susceptibility does not correlate with cAMP accumulation and ganglioside content.

The CT-mediated signaling mechanisms have been widely used as a tool for helping the knowledge of the more complex mechanisms regulating cell growth and proliferation in which gangliosides are involved as receptors and cAMP as second messenger. In the present study we compare the susceptibility of two murine cell lines (SR-4987 stromal cells and L1210 leukemic cells) to inhibitory effect of cholera toxin (CT) on cell growth and correlate their sensitivity to CT with ganglioside content and intracellular cAMP accumulation. The results indicate a very different response of the two cell lines to CT treatment. L1210 cells (which contain GM1a ganglioside) are sensitive to the inhibiting activity of CT (IC50 in the clonogenic assay = 10(-9) M) but no cAMP accumulation was observed after the treatment. SR-4987 cells (which lack GM1a) show a dramatic increase of intracellular cAMP without any inhibition of cell growth following the CT treatment until 10(-8) M. However, after SR4987 cells have incorporated GM1a they became susceptible to CT (with a IC50 value = 10(-11) M). The comparison of these results with our previous studies on WEHI-3B leukemia cells confirms the remarkable heterogeneity of cell sensitivity to the growth inhibition by CT by emphasizing that this inhibition is the final event of very different mechanisms in which CT binding to a specific ganglioside seems to be necessary and sufficient whereas cAMP accumulation may not be coupled with the antiproliferative effect of CT.

Expression of B cell markers on SR-4987 cells derived from murine bone marrow stroma.

The murine cell line SR-4987 was originated in our laboratory from adherent cells of a long term bone marrow culture. SR-4987 cells do not express p21-ras and c-fms products on membrane whereas secrete M-CSF, evidence a fibroblast-like morphology and are vimentine positive. This line shows a very poor "in vitro" agar clonogenicity which is not modulated by the addition of different cytokines and growth factors (M-CSF, GM-CSF, G-CSF, IL-3, IL-7, alpha-TNF, PDGF, and EGF). On the contrary, a dramatic increase in clonogenicity is observed in the presence of bFGF. The RT-PCR investigation evidences the mRNA encoding for bFGF, IL-7, GM-CSF, and SCF (c-kit ligand). The analysis of CD antigen expression on SR-4987 cell membrane indicates a phenotype (CD5+, CD44+, 45R(B220)+, sIg+, 5'-nucleotidase+) that is consistent with a B cell feature. Our observations suggest that exogenous bFGF might represent an appropriate stimulus for inducing the SR-4987 cells proliferation also in the absence of cell-substrate anchorage. Further, they indicate that SR-4987 cells could represent a particular differentiation stage in which characters of "stromal cell" and "B cell" are coexpressed in agreement with the hypothesis of a common stromal-hematopoietic differentiation.

The different inhibiting effect of cholera toxin on two leukemia cell lines does not correlate with their toxin binding capacity.

The murine leukemia cell lines L1210 and WEHI-3B show a very different sensitivity to the cholera toxin (CT). The in vitro growth of L1210 is completely inhibited by 10(-8) M CT, while WEHI-3B growth shows the same inhibition at 10(-11) M. The analysis of membrane ganglioside pattern of the two cell lines shows that in L1210 cells the major component is the GM1a ganglioside while the monosialogangl oside fraction from WEHI-3B is entirely composed of gangliosides of the 'b' series among which GM1b is the more represented. The total cholera toxin binding capacity of the ganglioside extract from L1210 cells is more than hundred fold higher than that of WEHI-3B and this difference is also confirmed by the number of CT receptors/cell and by the binding of FITC-B subunit of CT on the cells. These surprising data are in conflict with the poor sensitivity to CT evidenced by L1210 compared to WEHI-3B cells. In order to clarify this discrepancy we investigated the cAMP accumulation, the cell viability and the clonogenicity of these two leukemia cell lines following the treatment with CT and forskolin (FRSK). The treatment of WEHI-3B cells with CT induces a dramatic increase of intracellular cAMP which highly correlates with cell death and the decrease of clonogenicity and this result is partially obtained by the treatment with FRSK. L1210 cells do not evidence significant cAMP accumulation neither with CT nor with FRSK treatment. These data suggest that the different inhibiting effect of CT on WEHI-3B and L1210 cells does not correlate with their different pattern of gangliosides and the related toxin binding capacity. Further they indicate that the growth inhibition of WEHI-3B cells is closely related with a cAMP-dependent cell killing mechanism, while the inhibition of L1210 growth (produced by high concentration of CT) is mediated by a cAMP independent mechanism.

Loss of porins following carbapenem-resistance selection and adherence modification in enterobacteria.

The acquired resistance to the carbapenems is frequently joined to modified expression of porins or other outer membrane (OM) structures, thus bacterial adherence, that also depends on the presence of peculiar surface structures, might theoretically be influenced. In this study the ability to adhere to Hep-2 and I-407 eukaryotic cell monolayers was assayed for two susceptible strains of Serratia marcescens, one strain of Enterobacter cloacae and one of Providencia rettgeri in comparison with that of isogenic resistant mutants selected either by carbapenems or by cephalosporins. The mutants appeared slightly less adherent than the wild type strains, however, due to the high variability of this kind of assay, the differences observed in most cases could not be considered statistically significant. The data suggest that adherence, among the factors affecting the pathogenicity of the strains, remains probably unmodified in the resistant bacterial population possibly selected by a carbapenem treatment.

Different biological conditions influencing bacterial adherence assay.

Adherence of bacteria to animal cells is considered the first step in the pathogenesis of many infectious diseases. The most suitable techniques developed in vitro to check the capacity of bacteria to adhere to different tissues use monolayers of established cell lines. We studied the influence of incubation time (1, 2, 3 hours), cell substrates (Hep-2, H-407) and the number of bacteria per cell (1, 10, 100, 1000) on the adherence index (number of adherent bacteria per cell determined by microscopic count) of the fimbriated Escherichia coli 454 strain, Proteus rettgeri 25 and Enterobacter cloacae 10. The data were analyzed with different statistical methods and the results evidenced that all the conditions considered affect either the end-point of the test or the adherence index. Our observations indicate that the different methods used make it impracticable to compare many data from the literature and suggest the need to search for more homogeneity in this type of assay.

In vitro short-term and long-term cytotoxicity of fluoroquinolones on murine cell lines.

Short-term and long-term cytotoxicity of four fluoroquinolones (ciprofloxacin, rufloxacin, ofloxacin, lomefloxacin) on 7 established murine cell lines (WEHI-3B, L1210, EL4, P388D1, 32DC13, L929, SR-4987) by a microtiter MTT assay have been studied. In short-term cytotoxic test (24 hr), cell lines with a high proliferating cell rate (as leukemias) showed a greater sensitivity to quinolones than other cell lines. In long-term cytotoxic test (7 days) no different sensitivity was observed among the cell lines. In short-term test ciprofloxacin and rufloxacin were more toxic than lomefloxacin and ofloxacin whereas in the long-term test the activity of the four quinolones was similar. Ratio between IC50 on cell lines and MIC50 against gram negative bacteria evidenced remarkable differences when short-term or long-term cytotoxic tests were considered. The results confirm toxic activity of quinolones on mammalian cells evidencing that the sensitivity to quinolones, in short-term cytotoxic test, correlates with the doubling time of cell population. The results further suggest that long-term cytotoxic tests measure better the antiproliferating activity of quinolones providing a more powerful assay to investigate their in vitro toxicity.

A comparison between the hemolytic and antibacterial activities of new quaternary ammonium polymers.

New quaternary ammonium polymers, which in a previous work had shown relevant antibacterial properties, have been investigated as regards to their hemolytic activity (HA) in comparison with a low molecular weight commercial antibacterial agent, Steramine G (SG). All polymers exhibit negligible, or at most modest, HA at dosages and contact times at which SG is strongly hemolytic.

Lack of in vitro antiviral activity of fluoroquinolones against herpes simplex virus type 2.

The antiviral activity against herpes simplex virus type 2 (HSV-2) of five fluoroquinolones (ciprofloxacin, lomefloxacin, ofloxacin, pefloxacin, rufloxacin) was tested in vitro. Their efficacy was evaluated as reduction of the cytopathic effect (CPER) exerted by HSV-2 on Vero cells in comparison with novobiocin and acycloguanosine. Our results show a very poor antiviral effect of five quinolones (CPER50 = 200 mg/l) that was comparable with their cytotoxicity (TCIC50 less than 200 mg/l). Novobiocin shows a lower toxicity (TCIC50 = 400 mg/l) and a slight antiviral activity (CPER50 = 120 mg/l). Acycloguanosine shows a TCIC50 greater than 400 mg/l and a CPER50 of 3.125 mg/l. The therapeutic indices gave values ranging from 0.12 to 2 for quinolones, of 3.3 for novobiocin, and greater than 128 for acycloguanosine. The antiviral efficacy of acycloguanosine was not affected by concentrations of quinolones active against bacteria (1-10 mg/l) whereas it was drastically reduced by higher doses of quinolones (greater than 50 mg/l). Our data suggest that fluoroquinolones cannot be considered drugs able to inhibit HSV-2 replication in vitro.

Establishment and characterization of a new murine cell line (SR-4987) derived from marrow stromal cells.

A new murine cell line designated as SR-4987 was established by treating a long-term bone marrow culture with the supernatant from Y-1 cells which actively produce viral C-particles (MuLV). The line showed a fibroblast-like morphology and its mesodermal origin was confirmed by immunocytochemical staining. Flow cytometric analysis of DNA index evidenced a tetraploid number of chromosomes whereas cell cycle analysis showed 34.8% of cells in S phase and 60.7% in G1. In vitro growth studies demonstrated a population doubling time of 14.7 h, a good plating efficiency (52.3%) and a very poor agar clonogenic capacity (0.6%). SR-4987 was tumorigenic only in syngeneic mice in which sarcomas were induced. The line produced M-CSF in the culture supernatant whereas G-CSF, IL-3 and GM-CSF were not detected. Studies are in progress to assess the production of other cytokines and to verify if same autocrine growth factor is involved in the control of SR-4987 proliferation. Our line provides a further model of stromal cells for studying the interaction between hemopoietic progenitors and their microenvironment, as well as to study factors produced by stromal cells acting as modulators of proliferation and differentiation of related cell populations.

New quaternary ammonium polymers as antimicrobial agents. Part II. Alkylation products of linear aliphatic poly (aminodisulphides).

Two new polymeric disulphides containing t-amino groups in their main chain, namely poly[1,8-(3,6-dimethyl-3,6-diaza) octaine diyl disulphide] (5) and poly[1,8-(1,12-(3-10-dimethyl-3,10-diaza) dodecane diyl disulphide] (6) were prepared by the polyoxidation of 3,6-dimethyl-3,6-diazaoctane-1,8-dithiol (3) and 3,10-dimethyl-3,10-diazadodecane-1,12-dithiol (4), respectively. They were quaternized with methyl iodide and benzyl bromide, and the resulting quaternary ammonium polymers were preliminarily tested for antimicrobial activity against Escherichia coli K12, Pseudomonas aeruginosa, and Staphylococcus aureus. All the quaternized products showed interesting killing potency against P. aeruginosa. The benzylated products, besides being more active against P. aeruginosa, showed fair activity also against the other bacterial strains tested.

Inhibition of murine leukemia (WEHI-3B and L1210) proliferation by cholera toxin B subunit.

Cholera holotoxin produces both stimulation and inhibition of the growth of different cell populations. These opposite effects were both attributed to the enzymatic activity of the subunit A that activates adenylate cyclase, increasing the intracellular level of cAMP. We observed that the B subunit of cholera toxin produced by itself an inhibition of the 'in vitro' growth of two murine leukemia cell lines (L1210 limphoid leukemia and WEHI-3B myelomonocytic leukemia). The sensitivity of WEHI-3B cells towards cholera toxin was about 5000-times higher than that of the L1210 cells, whereas the two leukemias showed an identical sensitivity to the B subunit (IC50 = 5.10(-10) M for L1210 and 10(-10) M for WEHI-3B). The inhibition produced by the B subunit was neutralized by GM1 and in a minor degree by type II gangliosides. The two leukemias showed a remarkable difference in their gangliosides contents (L1210 cells contained GM1 (80.6%) and GM2 (19.4%), while WEHI-3B cells contained GM1 (28.2%), Fuc-GM1 (44.9%) and a band (26.9%) with a chromatographic mobility between GD1a and GD1b). The inhibition could be explained by a competitive mechanism between the B subunit and some autocrine factor binding GM1-containing receptors. Our data strengthen the suggestion to consider gangliosides as very important pleiotropic biomodulators.

Effect of fluoroquinolones on the in-vitro proliferation of myeloid precursor cells.

The effects of three fluoroquinolones (ofloxacin, pefloxacin and rufloxacin) on the in-vitro proliferation of murine myeloid cells were studied. Their activity was compared with that of nalidixic acid and novobiocin. Therapeutic concentrations of quinolones do not affect the physiological course of myelopoiesis. Only very high concentrations of drug (greater than 70 mg/l) affect bone marrow cell growth producing a dose-dependent inhibition. Because quinolones are active on topoisomerase II from eukaryotic cells they can modulate mammalian cell growth.