Colony-stimulating factor produced by murine adrenocortical tumor cells.

Conditioned medium obtained from the adrenocortical LAF1 mouse tumor (Y-1) cell cultures was able to stimulate the proliferation and the differentiation of granulocyte-macrophage precursors in the normal murine bone marrow. Colony-stimulating factor (CSF) was spontaneously produced by Y-1 cells also in serum-free cultures. By two cycles of gel chromatography on Sephadex G-150 of concentrated conditioned medium two peaks of colony-stimulating activity were isolated that corresponded to apparent molecular radii of 100,000 and 29,000, respectively. At this step of purification, the two factors gave a similar dose-response curve, showed a remarkable resistance to the heat treatment and pH changes, and were not extracted by ether. Because Y-1 cells resulted in infection by retrovirus, they provide a useful model to investigate the relations between viral coded information and CSF production.

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.

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.

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.

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.

Inhibition of granulocytic-macrophagic precursor cells (CFU-C) by heat-labile enterotoxin (LT) produced by Escherichia coli.

Enterotoxigenic strains of Escherichia coli cause diarrhea by production of heat-labile enterotoxin (LT) which acts through stimulation of membrane-bound adenylate-cyclase in epithelial cells. We studied in vitro production of LT by growing E. coli H 10407 in different synthetic media in comparison with Penassay broth. Non-toxigenic E. coli K12 was used as control. We obtained positive response in Y-1 cell assay for LT activity with all filtrates from E. coli H 10407 cultures. These filtrates inhibit 3H-thymidine uptake by Ehrlich Ascites Carcinoma (EAC) cells and the proliferation of granulocytic-macrophagic precursors (CFU-C) in murine bone marrow. Filtrates did not stimulate CFU-C in absence of CSF. Heat-treated (121 degrees C for 30 minutes) and dialyzed (molecular cut 15,000 daltons) filtrates lost their cytotoxicity against Y-1 cells maintaining the inhibitory activity on CFU-C proliferation. This phenomenon may be regarded as the result of a competitive mechanism between LT and CSF (Colony Stimulating Factor) on the receptor system of committed stem cells.

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.

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.

Isolation and partial characterization of temperature bacteriophages from enteropathogenic strains of Escherichia coli 0111:K58.

We report studies on ten strains of Escherichia coli 0111:K58 isolated from children with acute diarrhea. Our results show that these E. coli strains do not produce the pathogenic factors of enterotoxic E. coli (ETEC) and are lysogenic for phages belonging to two groups that differ for host range, kinetics of thermal inactivation, antigenicity and morphology. These data support the hypothesis that these phages may in vivo contribute to reduction of the number of common E. coli strains by lytic infection favouring the development of the enteropathogenic strain of E. coli.

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.

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.

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.

Production of colony stimulating factor by EL-4 lymphoma cells stimulated with phytohemoagglutinin.

Murine EL-4 lymphoma cells when stimulated with optimal doses of phytohemoagglutinin (PHA) release colony stimulating factor (CSF) in the conditioned medium. CSF production depends on PHA and stops when the lectin is withdrawn. The production is dependent on protein synthesis and is not related with DNA replication. The CSF produced by El-4 cells stimulates murine bone marrow cells to differentiate in vitro into mature granulocytes. Unstimulated EL-4 cells do not produce CSF while its conditioned medium is able to inhibit EL-4 proliferation in vitro. Gel filtration chromatography of conditioned medium from PHA-stimulated EL4 cells yields a peak of activity corresponding to an apparent molecular weight of 20,000-25,000 daltons.

Isolation of a colony-stimulating factor produced by L 1210 murine leukemia cells.

Murine L1210 leukemia cells spontaneously produce very low amounts of colony stimulating factor (CSF). CSF production was markedly increased by stimulating L1210 cells with lipopolysaccharide, lectins, and sheep red blood cells. From the conditioned medium of phytohemagglutinin-stimulated L1210 cells we isolated a CSF with an apparent molecular weight of approximately 27,000. This CSF promoted the proliferation and the differentiation of murine GM-CFU showing a weak differentiation-inducing activity on WEHI-3 D (+) cells.

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.

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.