Ilario Viano

Macrophage Stimulating Protein (MSP) evokes superoxide anion production by human macrophages of different origin

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. 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. 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. 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.

Pt(II) complexes with different N-donor aromatic ligands for specific inhibition of telomerase

Abstract A number of cis -dichloroplatinum(II) derivatives containing aromatic N-donor ligands such as pyridine and/or 5-substituted isoquinolines have been synthesized and fully characterized, with particular use of Raman spectroscopy and mass spectrometry. These complexes have been tested for specific inhibition of telomerase, which could represent a selective target for cancer chemotherapy. The screening of such potential drugs has been performed by a biochemical assay on the isolated enzyme. It is noteworthy that the cis -[PtCl 2 (pyridine)(5-SO 3 H-isoquinoline)] compound exhibited remarkable telomerase inhibition at a concentration as low as 10 −7 M.

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.

Lymphokine production in mouse mixed lymphocyte reaction (mlr). II. Tentative mapping of murine alloantigens activating migration inhib- ition factor and interferon release and their relationship with those activating proliferative response.

We have investigated alloantigen differences which stimulate lymphokine release and3H-TdR uptake in primary ‘one-way’ MLC among allogeneic mice. When mice differing at the wholeH-2 region were tested, MIF and immune IF release was observed, along with a marked3H-TdR uptake. Differences atK, D, orI-S-G regions stimulate both lymphokine release and3H-TdR uptake, though stronger immune IF and3H-TdR responses were observed with differences atI-S-G regions. On the other hand, when mice differing in their minor histocompatibility antigens, and notably at theMls locus, were tested, lymphokine release took place even in the absence of proliferation. Lastly, in MLC between mice differing at multiple minor loci, butH-2 andMls matched, MIF release only, and not immune IF and3H-TdR responses were observed in a few combinations. These findings show that T lymphocytes can recognize alloantigens by releasing lymphokines even without going through proliferation. Moreover, different levels of T-lymphocyte activation exist, depending on the kind of stimulating alloantigens present.

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.

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.

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.

Hemodynamic effects of the intravenous administration of cyclorirobuxine D in anesthetized pigs

Abstract The present study was undertaken in anesthetized pigs to determine the primary effects of cyclorirobuxine D given intravenously on hemodynamic variables. In eight pigs, the administration of 1.5 mg/kg of cyclorirobuxine D 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 dt max ) was observed. In four pigs, the decrease in heart rate and the increase in left ventricular dP dt max were progressively augmented by graded increases in the dose of cyclorirobuxine D. In six pigs, the responses of hemodynamic variables to cyclorirobuxine D were not affected by blockade of cholinergic and adrenergic receptors. In a further six pigs, blockade of nitric oxide synthase with Nϖ-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 dt max and aortic blood pressure. The present study showed that intravenous administration of cyclorirobuxine D 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.

Development, replication, and reversion of mecillinam-induced ovoid form ofSalmonella typhimurium

Concentrations of mecillinam ranging from 0.8 to 0.0125 μg/ml in Trypticase soy broth causedSalmonella typhimurium to assume an ovoid morphology. The presence of chloramphenicol or the absence of nutrients or nonphysiological temperatures prevented the change to ovoid morphology, whereas the presence of nalidixic acid modified the effect of mecillinam. Ovoid forms replicated as ovoids. No intermediate rod morphology was evident during eight daily subculturings of ovoids in mecillinam. Reversion of ovoids to normal morphology occurred within 3 h in drug-free medium, or within 15 min after the addition of penicillinase. Inhibition of either DNA or protein synthesis, or the absence of nutrients, or incubation in nonphysiological temperatures prevented reversion of ovoids to normal morphology.