Macrophage stimulating protein (MSP) evokes superoxide anion production by human macrophages of different origin.

1. Macrophage Stimulating Protein (MSP), a serum factor related to Hepatocyte Growth Factor, was originally discovered to stimulate chemotaxis of murine resident peritoneal macrophages. MSP is the ligand for Ron, a member of the Met subfamily of tyrosine kinase receptors. The effects of MSP on human macrophages and the role played in human pathophysiology have long been elusive. 2. We show here that human recombinant MSP (hrMSP) evokes a dose-dependent superoxide anion production in human alveolar and peritoneal macrophages as well as in monocyte-derived macrophages, but not in circulating human monocytes. Consistently, the mature Ron protein is expressed by the MSP responsive cells but not by the unresponsive monocytes. The respiratory burst evoked by hrMSP is quantitatively higher than the one induced by N-formylmethionyl-leucyl-phenylalanine and similar to phorbol myristate acetate-evoked one. 3. To investigate the mechanisms involved in NADPH oxidase activation, leading to superoxide anion production, different signal transduction inhibitors were used. By using the non selective tyrosine kinase inhibitor genistein, the selective c-Src inhibitor PP1, the tyrosine phosphatase inhibitor sodium orthovanadate, the phosphatidylinositol 3-kinase inhibitor wortmannin, the p38 inhibitor SB203580, the MEK inhibitor PD098059, we demonstrate that hrMSP-evoked superoxide production is mediated by tyrosine kinase activity, requires the activation of Src but not of PI 3-kinase. We also show that MAP kinase and p38 signalling pathways are involved. 4. These results clearly indicate that hrMSP induces the respiratory burst in human macrophages but not in monocytes, suggesting for the MSP/Ron complex a role of activator as well as of possible marker for human mature macrophages.

Decreased drug accumulation and increased tolerance to DNA damage in tumor cells with a low level of cisplatin resistance.

In an attempt to examine the cellular changes associated with cisplatin resistance, we selected a cisplatin-resistant (A43 1/Pt) human cervix squamous cell carcinoma cell line following continuous in vitro drug exposure. The resistant subline was characterized by a 2.5-fold degree of resistance. In particular, we investigated the expression of cellular defence systems and other cellular factors probably involved in dealing with cisplatin-induced DNA damage. Resistant cells exhibited decreased platinum accumulation and reduced levels of DNA-bound platinum and interstrand cross-link frequency after short-term drug exposure. Analysis of the effect of cisplatin on cell cycle progression revealed a cisplatin-induced G2M arrest in sensitive and resistant cells. Interestingly, a slowdown in S-phase transit was found in A431/Pt cells. A comparison of the ability of sensitive and resistant cells to repair drug-induced DNA damage suggested that resistant cells were able to tolerate higher levels of cisplatin-induced DNA damage than their parental counterparts. Analysis of the expression of proteins involved in DNA mismatch repair showed a decreased level of MSH2 in resistant cells. Since MSH2 seems to be involved in recognition of drug-induced DNA damage, this change may account for the increased tolerance to DNA damage observed in the resistant subline. In conclusion, the involvement of accumulation defects and the increased tolerance to cisplatin-induced DNA damage in these cisplatin-resistant cells support the notion that multiple changes contribute to confer a low level of cisplatin resistance.

Tachykinin receptors on human monocytes: their involvement in rheumatoid arthritis.

Three types of tachykinin receptors, namely NK1, NK2 and NK3, are known to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), respectively. Experimental evidence indicates that SP and NKA modulate the activity of inflammatory and immune cells, including mononuclear ones. This study evaluated the effects of mammalian tachykinins and selective tachykinin agonists and antagonists on human monocytes isolated from healthy donors: SP, NKA and NKB all evoked a dose-dependent superoxide anion (O2-) production and the NK2 selective agonist [beta-Ala8]-NKA(4-10) induced a full response. The NK3 selective agonist senktide was inactive, while the NK1 selective agonists septide and [Sar9Met(O2)11]SP displayed some effects. These results indicate that NK2 and also some NK1 receptors are present in monocytes isolated from healthy donors. The role of tachykinin receptor activation in rheumatoid arthritis was also investigated, by measuring O2- production and TNF-alpha mRNA expression in monocytes isolated from rheumatoid patients. Tachykinins enhanced the expression of this cytokine in both control and rheumatoid monocytes and NK2 receptor stimulation was shown to trigger an enhanced respiratory burst in monocytes from rheumatoid patients. In conclusion, these results indicate that NK2 and NK1 receptors are present on human monocytes, the former being preferentially involved in rheumatoid arthritis.

Modulation by protein kinase C of the enhanced responsiveness to tachykinins in ovalbumin-sensitized guinea pig alveolar macrophages.

As previously reported, alveolar macrophages (AMs) from ovalbumin-sensitized guinea pigs present an enhanced responsiveness to tachykinins but not to N-formylmethionyl-leucyl-phenylalanine (fMLP). We have investigated the biochemical mechanisms underlying this varied responsiveness to tachykinins. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) induced a larger superoxide anion (O2-) production in AMs from sensitized guinea pigs, as did tachykinins. Pretreatment of AMs with pertussis toxin abolished tachykinin-evoked respiratory burst, had no effect on PMA-evoked O2- production and strongly inhibited fMLP-evoked one, with no appreciable variation between control or sensitized AMs. Staurosporine and its derivative cgp 41251, significantly decreased PMA- and tachykinin-evoked O2- production in both populations, being more potent in control AMs, but exerted little effects against fMLP. Pretreatment of AMs with PMA significantly inhibited fMLP-, PMA- and tachykinin-evoked O2- production in both control and sensitized AMs. fMLP, substance P (SP), neurokinin A (NKA) and the NK2 agonist [beta-Ala8]-NKA(4-10) dose-dependently increased [3H] phorbol 12, 13 dibutyrate (PDBu) binding to control and sensitized AMs. While fMLP exerted similar effects in both populations, dose-response curves for SP1 NKA and the NK2 receptor agonist were shifted leftwards (1, 4 and 3 orders of magnitude, respectively) in sensitized AMs. These results indicate a possible PKC involvement in the enhanced responsiveness to tachykinins in actively sensitized AMs.

Tachykinin activation of human monocytes from patients with rheumatoid arthritis: in vitro and ex-vivo effects of cyclosporin A.

Three types of tachykinin receptors, namely NK1, NK2 and NK3, are known to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), respectively. We previously demonstrated that NK1 and NK2 receptors are present on human monocytes, SP and NKA inducing superoxide anion production and tumor necrosis factor-alpha (TNF-alpha) mRNA expression. NK2 receptor stimulation also triggered an enhanced respiratory burst in monocytes isolated from rheumatoid arthritis (RA) patients. This study was aimed to evaluate the in vitro and ex-vivo effects of cyclosporin A (CsA) on tachykinins-evoked TNF-alpha release from monocytes of healthy donors and RA patients. CsA (100 ng/ml) potently inhibited phorbol ester- and tachykinin-evoked TNF-alpha secretion. In RA patients treated with CsA (Sandimmun Neoral 2.5 mg/kg/day, a significant time-dependent reduction in TNF-alpha secretion from monocytes was measured. This may contribute to the CsA therapeutic activity in RA.

Tachykinin activation of human alveolar macrophages in tobacco smoke and sarcoidosis: a phenotypical and functional study.

Substance P (SP) and neurokinin A (NKA), which exert bronchoconstrictor effects on human airways, are known to interact with inflammatory and immune cells, including monocyte macrophages. We have evaluated the effects of SP, NKA and the NK2 selective agonist [beta-Ala8]-NKA(4-10) on alveolar macrophages (AM) isolated from 4 healthy smokers and 4 non-smoker active pulmonary sarcoid patients. An accumulation of activated mononuclear phagocytes, as well as elevated angiotensin-converting enzyme (ACE) activity, has been evidenced in both clinical conditions. The phenotype of AMs in the studied subjects was characterized by an elevated expression of CD68+, HLA-DR+ and CD14+, CD14+ being significantly less in sarcoidosis as compared to smokers. SP, NKA and the NK2 selective agonist evoked superoxide anion (O2-) production in AMs obtained from sarcoid patients or healthy smokers. While SP acted in a non-dose-dependent manner in both conditions, NKA and [beta-Ala8]-NKA(4-10) evoked a dose-dependent respiratory burst (ED50 = 0.25 and 0.26 nM, respectively) in smokers, but not in sarcoidosis. The more marked phenotypical expression correlated well with the ability of NK2 receptors to activate AMs in smoker subjects.

Tachykinin activation of human monocytes from patients with interstitial lung disease, healthy smokers or healthy volunteers.

Three types of tachykinin receptors, NK(1), NK(2)and NK(3), have been described to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) respectively. Experimental evidence indicates that SP and NKA modulate the activity of inflammatory and immune cells, including mononuclear ones, and points to their involvement in lung pathophysiology. We previously reported that NK(1)and NK(2)receptors are present on monocytes (MO) isolated from healthy donors or rheumatoid patients - a greater sensitivity to NK(2)receptor stimulation was observed in the latter condition. This study evaluated the effects of SP and NKA, as well as NK(1)and NK(2)selective agonists and antagonists, on MO obtained from healthy volunteers, healthy smokers or patients with interstitial lung diseases (e.g. sarcoidosis and idiopathic pulmonary fibrosis). Superoxide anion (O(2)(-)) production was chosen as a parameter of cell activation. SP and NKA dose-dependently evoked O(2)(-)production from MO in all the conditions evaluated, their effects being competitively antagonized by selective antagonists (CP 96 345 and MEN 10 627, respectively). When selective NK(1)and NK(2)agonists were used, [Sar(9)Met(O(2))(11)]SP, a selective NK(1)agonist, induced a more than doubled O(2)production in MO obtained from patients with interstitial lung diseases as compared to healthy volunteers, whereas MO isolated from healthy volunteers were more sensitive to NK(2)receptor stimulation.

Adenosine modulation of primed human neutrophils.

Human neutrophils have been demonstrated to possess both adenosine A1 and A2 receptors: activation of adenosine A2 receptors inhibits the respiratory burst, assayed as superoxide anion production (O-2) from cells stimulated by the bacterial peptide N-formylmethionyl-leucyl-phenylalanine (FMLP). Exposure of neutrophils to different combinations of stimuli results in synergistic or primed responses. These responses can be measured by challenging the cells either with a combination of FMLP and platelet activating factor (PAF), or with a combination of PAF and the neuropeptide substance P, which by itself does not induce O-2 production. In order to evaluate the ability of adenosine receptor agonists to inhibit O-2 production by primed or synergistically stimulated neutrophils, a non-selective adenosine receptor agonist, 2-chloroadenosine, was tested in comparison with reportedly selective ligands of adenosine A1 and A2 receptor types, N6-cyclopentyladenosine (CPA) and 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethyl-carboxamido adenosine (CGS 21680). The order of activity CGS 21680 > 2-chloroadenosine > CPA indicates that adenosine A2 receptors mediate the inhibition of the respiratory burst even when neutrophils are primed or synergistically activated. 8-Phenyltheophylline antagonized the effects of these adenosine receptor agonists in a competitive way.

Coronary effects of cyclovirobuxine D in anesthetized pigs and in isolated porcine coronary arteries.

The present study was undertaken in anesthetized pigs and in isolated porcine coronary arteries to determine the primary coronary effects of cyclovirobuxine D. In six pigs, the intravenous administration of 1.5 mg/kg of cyclovirobuxine D whilst preventing changes in heart rate and aortic blood pressure caused increases in left ventricular dP/dtmax and coronary blood flow which respectively averaged 10% and 23.9%. These responses were progressively augmented by graded increases in the dose of the drug (four pigs) and were not affected by blockade of cholinergic and adrenergic receptors (five pigs). Intravenous blockade of nitric oxide synthase (L-NAME, five pigs) abolished both responses, while intracoronary injection of L-NAME (five pigs) abolished only the coronary vasodilatation. In ten isolated coronary segments, cyclovirobuxine D significantly reduced the degree of potassium chloride-induced contraction. This reduction was not affected by inhibition of cyclooxygenase with indomethacin (five segments) or potassium channels blockade with glibenclamide (five segments), but it was abolished by L-NAME (five segments) or removal of endothelium (five segments). The present study showed that cyclovirobuxine D caused a primary effect of coronary vasodilatation, which involved mechanisms related to the endothelial release of nitric oxide.

Hemodynamic effects of the intravenous administration of cyclovirobuxine D [correction of cyclorirobuxine D] in anesthetized pigs.

The present study was undertaken in anesthetized pigs to determine the primary effects of cyclovirobuxine D [corrected] given intravenously on hemodynamic variables. In eight pigs, the administration of 1.5 mg/kg of cyclovirobuxine D [corrected] caused a small increase in aortic blood pressure. When this response was prevented, a decrease in heart rate was obtained in each of the eight pigs. When this response was also prevented, an increase in the maximum rate of change of left ventricular systolic pressure (left ventricular dP/dtmax) was observed. In four pigs, the decrease in heart rate and the increase in left ventricular dP/dtmax were progressively augmented by graded increases in the dose of cyclovirobuxine D [corrected]. In six pigs, the responses of hemodynamic variables to cyclovirobuxine D [corrected] were not affected by blockade of cholinergic and adrenergic receptors. In a further six pigs, blockade of nitric oxide synthase with N omega-nitro-L-arginine methyl ester did not affect the decrease in heart rate caused by the drug, but abolished the increases in left ventricular dP/dtmax and aortic blood pressure. The present study showed that intravenous administration of cyclovirobuxine D [corrected] primarily caused a decrease in heart rate and an increase in left ventricular inotropic state, which secondarily determined an increase in aortic blood pressure, and suggested that the response of heart rate involved a direct effect of the drug on the heart, while the response of left ventricular contractility was related to mechanisms dependent on the release of nitric oxide.

Positive interaction between interferon and bleomycin in vitro and in animal experimental models.

Effect of combined bleomycin and interferon on cell growth was evaluated in vitro, on 2 leukemia cell lines, and on mice bearing a murine leukemia. Human alpha-A interferon or murine alpha-beta interferon were used. Synergism was observed in both models and the sequence of administration was decisive in this respect. The in vivo results were not completely comparable with those observed in vitro. This finding suggests that the in vitro results reflect the direct effects of the drugs on tumor cells, meanwhile the interaction between drugs in vivo are affected by pharmacokinetic characteristics and by host defense systems.

Drug sensitivity tests after cell separation on density gradients.

Cells from two leukaemic cell lines (K562 and L1210) were separated on a discontinuous Percoll gradient with densities from 1040 to 1090 by centrifugation. The cells recovered from gradient interfaces were checked for their DNA content and subjected to two different drug sensitivity tests using three cytostatic drugs. The purpose of the study was to compare the sensitivity of a sample of the whole population with that of the fractions thus obtained. Depending on the test used and the line tested, there was a 5.5 to 55.5% discordance between the results for the fractions and the non-separated cells.

Phase II study of alpha-interferon plus bleomycin in advanced non-small cell lung cancer.

A synergistic effect between alpha-Interferon and Bleomycin has been recently shown in in vitro and in vivo experimental systems. Although active, Bleomycin and other antineoplastic drugs give low response rates in non-small cell lung cancer, but, like the other antineoplastic agents, responses are short-lived. We treated 13 patients with advanced non-small cell lung cancer with the combination Bleomycin 15 mg/m2 i.v. at hour 0 and alpha-Interferon 9 X 10(6) U i.m. given at hours 6, 30 and 54. Major side effects were pyrexia, astenia and anorexia; only one case of moderate leukopenia was observed. No major responses were obtained and stable disease lasted a median of 5 months. Further study of this combination is not warranted in patients with pretreated non-small cell lung cancer.

New anthracenedione derivatives: interaction with DNA and biological effects.

Two anthracenedione derivatives [1 - (omega - diethylaminopropylamido) - 4 - hydroxy - 9,10 - anthracenedione hydrochloride (I) and 1 - (omega - diethylaminopropylamido) - 2 - methoxy - 4 - hydroxy - 9, 10 - anthracenedione hydrochloride (II)], having an electron-rich planar chromophore and an amino-substituted side chain, have been synthesized. Their binding ability to DNA was investigated by means of spectroscopic, equilibrium dialysis and fluorescence measurements. Their inhibition efficiency on nucleic acid synthesis was also evaluated both in mouse and human cells. Our results indicate that, in comparison with adriamycin, compound I shows a slightly weaker complexation ability to DNA, while compound II interacts with DNA at a substantially lower level. These data match quite well with the biological response on the inhibition of DNA and RNA synthesis exhibited by the above mentioned compounds; in fact compound I is slightly less efficient than adriamycin and about ten times more efficient than compound II. The close relationship between the results of physicochemical and biological studies is discussed.

Induction of 2'-5' oligoadenylate synthetase and activation of ribonuclease in tamoxifen treated human breast cancer cell lines.

In the present study the intracellular activity of oligoadenylate synthetase and ribonuclease have been evaluated in two human breast cancer cell lines treated with tamoxifen, a well known antiestrogenic drug. Increased levels of oligoadenylate synthetase and enhanced ribonuclease activity have been found in the cultures of CG5 cell line treated with concentrations of tamoxifen inhibiting cell growth. In the experiments with the EVSA-T cell line we found neither an antiproliferative effect nor an increased oligoadenylate synthetase and ribonuclease activity. It is likely that these enzymes are involved in the mechanisms by which this drug acts as an antiproliferative agent.

Modulation of neutrophil functions by a beta-interferon of human origin.

The effects of a beta interferon (beta-IFN) of human origin on different parameters of human neutrophil functioning were evaluated in vitro. In the concentration range 10(2)-10(4) IU/ml beta-IFN enhanced superoxide anion (O2-) production evoked by the peptide N-formylmethionyl-leucylphenylalanine (FMLP), 10(-7) M, when O2- production elicited by FMLP in the absence of beta-IFN treatment was 2.43 +/- 0.32 nmol cytochrome C reduced/10(6) cells/min. The enhancement afforded by 10(3) and 10(4) IU/ml beta-IFN was statistically significant. When FMLP-induced O2- generation was 4.55 +/- 0.3 nmol cytochrome C reduced/10(6) cells/min, no increase was detected after beta-IFN treatment. Phagocytosis was enhanced by beta-IFN in one case, with no effect in four others. Chemotaxis was not affected by exposure to beta-IFN. These results indicated that beta-IFN could exert modulating effects on some neutrophil functions that varied according to the extent of cell response to the stimuli.

Pertechnetate release from a water/oil microemulsion and an aqueous solution after subcutaneous injection in rabbits.

A water-oil microemulsion and an aqueous solution, both carrying pertechnetate, were injected subcutaneously in rabbits; release was observed by imaging the administration sites with a gamma-camera. Disappearance from the injection site of pertechnetate in aqueous solution was about ten times faster than that of pertechnetate in a microemulsion.

Toxic effects on rabbit kidney cell cultures of a new aminoglycoside.

In this study the cellular toxicity of two aminoglycosides (gentamicin and dactimicin) have been evaluated using rabbit kidney cell cultures. The correlation between toxic effect and antibiotic concentration, and between toxic effect and the period of contact have been evaluated. The toxic effect was measured in rabbit kidney cell cultures by means of the release of N-acetyl-beta-glucosaminidase. This enzyme was chosen in order to establish a correlation between in vitro and in vivo results. Gentamicin clearly shows in both tests a non-linear correlation between cellular toxicity and concentration or period of contact. Dactimicin, a new aminoglycoside probably less toxic than others, also shows in both tests a non-linear correlation between cellular toxicity and concentration or period of contact, but the release of enzyme was significantly reduced during the treatment of the cell cultures with this aminoglycoside.

Biological properties of benzodifuran derivatives.

The antiviral activity and the effect on DNA synthesis of two benzodifuran compounds were studied. DNA and some RNA viruses were significantly inhibited by concentrations ranging from 15 to 30 nM/ml. The inhibition of DNA synthesis in host cells was obtained with concentrations higher than those inhibiting virus replication. A favourable ratio between antiviral activity and inhibition of DNA synthesis of the host cells is present in these compounds. This activity is substantially due to the ability of the compounds to complex with DNA.

Excretion of aminoglycosides in a rat kidney model.

Dactimicin is a new aminoglycoside antibiotic with an interesting low nephrotoxicity in animal experimental models. In order to establish if a correlation exists between the nephrotoxic effect and renal pharmacokinetic behaviour of aminoglycosides, tobramycin, sisomicin and dactimicin were studied in a model of isolated and perfused rat kidney. Concentrations in venous and urinary effluent during a continuous perfusion of a constant concentration of drugs were evaluated. The influence of active transport was studied comparing excretion data obtained with perfusion buffer with or without glucose. In the present experimental model the renal excretions of the aminoglycosides tested are quite similar and cannot account for the different nephrotoxicity observed in animals.