Macrophage expression of interleukin-10 is a prognostic factor in nonsmall cell lung cancer.

Interleukin (IL)-10 is expressed in many solid tumours and plays an ambiguous role in controlling cancer growth and metastasis. In order to determine whether IL-10 is involved in tumour progression and prognosis in nonsmall cell lung cancer (NSCLC), IL-10 expression in tumour cells and tumour-associated macrophages (TAMs) and its associations, if any, with clinicopathological features were investigated. Paraffin-embedded sections of surgical specimens obtained from 50 patients who had undergone surgery for NSCLC were immunostained with an antibody directed against IL-10. TAMs and tumour cells positive for IL-10 were subsequently quantified. IL-10-positive TAM percentage was higher in patients with stage II, III and IV NSCLC, and in those with lymph node metastases compared with patients with stage I NSCLC. High IL-10 expression by TAMs was a significant independent predictor of advanced tumour stage, and thus was associated with worse overall survival. Conversely, IL-10 expression by tumour cells did not differ between stages II, III and IV and stage I NSCLC. In conclusion, interleukin-10 expression by tumour-associated macrophages, but not by tumour cells, may play a role in the progression and prognosis of nonsmall cell lung cancer. These results may be useful in the development of novel approaches for anticancer treatments.

[Occupational exposure to respiratory irritants and chronic obstructive pulmonary disease]. / Esposizione professionale ad agenti irritanti e sviluppo di broncopneumopatia cronica ostruttiva (BPCO).

Cigarette smoking and occupational exposure to respiratory irritants are the major riskfactors for chronic obstructive pulmonary disease (COPD), which is characterized by small-airway obstruction and destruction of pulmonary parenchyma: emphysema. We studied two groups of subjects: one exposed and the other one not-exposed to respiratory irritants, to investigate the relationship, if any, between occupational exposure and COPD. Subjects underwent high-resolution computed tomography-density mask of the chest to quantify pulmonary emphysema, pulmonary function tests, sputum induction and analysis for cell counts and measurements of metalloproteinase (MMP)-9 and its tissue inhibitor TIMP-1. Subjects with occupational exposure to respiratory irritants had higher residual volume and functional residual capacity, higher total inflammatory cells and neutrophils in induced sputum. By contrast, sputum levels of MMP-9, TIMP-1 and MMP-91TIMP-1 ratio did not differ between the 2 groups. We conclude that sputum induction and analysis could be a useful and non-invasive tool to study and follow subjects with occupational exposure to respiratory irritants.

[Substance P in the sputum of patients with chronic obstructive pulmonary disease and occupational exposure to respiratory irritants]. / Sostanza P nell'escreato di pazienti con broncopneumopatia cronica ostruttiva ed esposizione professionale ad irritanti respiratori.

The role of tachykininis in airway inflammation has been extensively demonstrated in experimental animal models, but evidence in humans is very sparse. The aim of this study was first to quantify the content of substance P (SP) in sputum of a group of patients, with chronic obstructive pulmonary disease and with exposure to occupational irritants. Secondly, to compare them with sputum SP content of a group of control subjects.

Pharmacokinetics of oxatomide in preterm infants.

The pharmacokinetics and tolerability of oxatomide oral suspension were investigated in preterm infants to evaluate the feasibility of planning a further study to assess its antiinflammatory effects and its effectiveness in preventing chronic lung disease (CLD). Following the administration of oxatomide 1 mg/kg, the peak plasma concentration (Cmax), the elimination half-life (t1/2), the volume of distribution (Vd), and the area under the curve (AUC) 0-36 h were measured and the following results were obtained: 42.2 +/- 15 ng/ml at 2 h after oxatomide administration, 41.4 +/- 2.0 h, 37.4 +/- 4.2 l/kg, and 468 +/- 52 ng/ml/h, respectively. Our study, therefore, demonstrated that a dose of 1 mg/kg/day oxatomide was effective in reaching therapeutic plasma levels in preterm infants without inducing adverse effects.

Ovalbumin sensitization of guinea-pigs reduces fMLP-induced calcium signal in alveolar macrophages.

In this study we analyzed the N-formyl-Met-Leu-Phe (fMLP)-induced calcium signal in alveolar macrophages (AM) isolated from ovalbumin-sensitized (OA-sensitized AM) and naive (naive AM) guinea-pigs. Guinea-pigs were sensitized by subcutaneous injection of OA and AM were isolated by bronchoalveolar lavage 6 weeks thereafter. On the following day, we measured in resting and fMLP-stimulated cells: intracellular calcium concentration by fura-2 imaging analysis, forskolin-induced cyclic AMP production and superoxide dismutase inhibitable superoxide anion release of adherent AM. Resting calcium was 82+/-5.0 nM (n=217) and 144+/-9.3 nM (n=213, P<0.001) in naive and OA-sensitized AM respectively. fMLP (10(-11)-10(-7)M) induced a dose-dependent calcium increase, 10(-8)M being the maximal effective dose in both naive and OA-sensitized AM. However, at all doses tested, this fMLP effect was lower in OA-sensitized than in naive AM. While in resting condition 10(-5)M forskolin increased cyclic AMP both in naive and OA-sensitized AM, in fMPL-stimulated AM forskolin was effective only in OA-sensitized AM. Superoxide anion release measured 10 min after fMLP stimulus was higher in naive than in sensitized AM. These data suggest that the fMLP-induced intracellular signal is different in OA-sensitized AM compared to naive cells.

Guanosine 3': 5'-cyclic monophosphate-dependent pathway alterations in ventricular cardiomyocytes of spontaneously hypertensive rats.

1. We investigated the effect of the NO-donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) on cardiomyocytes isolated from control normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 2. Ventricular cardiomyocytes were isolated from SHR and WKY hearts and imaging analysis of fura-2-loaded cells was performed in order to evaluate calcium transient in electrical field paced (0.5 Hz) cells. 3. In WKY cardiomyocytes, 1 - 200 microM SNAP dose-dependently increased cyclic GMP content. In basal conditions, cyclic GMP content of SHR cardiomyocytes was significantly higher than in WKY, but SNAP failed to further increase cyclic GMP over the basal level. 4. In control conditions, the Delta F/F and decay time of the calcium transient were similar in both strains. In WKY cardiomyocytes, SNAP (1 - 100 microM) reduced the decay time. In SHR cardiomyocytes, SNAP was ineffective. Dibutyryl cyclic GMP (10(-6) - 10(-8) M), a membrane permeable cyclic GMP analogue, behaved similarly to SNAP. 5. In WKY and SHR cardiomyocytes, 10(-8) M isoprenaline similarly increased Delta F/F and decreased the decay time. SNAP and dibutyryl cyclic GMP prevented the effect of isoprenaline in WKY, whereas both molecules were ineffective in SHR cardiomyocytes. In WKY, SNAP effects were blocked by pretreating cells with the cGK inhibitor KT-5823. 6. Western blotting analysis of cGK type I showed that the enzyme was expressed in WKY isolated cardiomyocytes, but absent in four out of five SHR preparations. 7. We concluded that the low expression of cGKI may determine the lack of NO/cyclic GMP-dependent regulation on calcium transient in SHR cardiomyocytes. This alteration may contribute to the development of heart hypertrophy in hypertensive status.

Mechanical stretch reveals different components of endothelial-mediated vascular tone in rat aortic strips.

1. Since the role of mechanical stretches in vascular tone regulation is poorly understood, we studied how stretch can influence endothelial tone. 2. Isometric contractions of isolated rat aortic helical strips were recorded. The resting tension was set at 0.7 g, 1.2 g or 2.5 g. Endothelium-preserved strips were precontracted with either phenylephrine or prostaglandin F(2 alpha) (PGF(2 alpha)). 3. In control conditions, acetylcholine (ACh) dose-dependently relaxed phenylephrine-precontracted strips independently of resting tension. 4. At 0.7 g resting tension, nitric oxide synthase (NOS) inhibitors did not reduce ACh-induced relaxation, while either a guanylyl cyclase inhibitor or a NO trapping agent prevented it. At 1.2 g and 2.5 g resting tensions, NOS inhibitors shifted the ACh dose-response curve to the right. 5. After preincubation with indomethacin (5 microM) or ibuprofen (10 and 100 microM), at 0.7 g and 1.2 g resting tensions, ACh induced an endothelium-dependent, dose-dependent contraction. ACh (10(-6) M) increased the contraction up to two times greater the phenylephrine-induced one. Lipoxygenase inhibitors prevented it. At high stretch, the ACh vasorelaxant effect was marginally influenced by cyclooxygenase (COX) inhibition. Similar results were obtained when aortic strips were precontracted with PGF(2 alpha). 6. Our data indicate that when resting tension is low, ACh mobilizes a stored NO pool that, synergistically with COX-derived metabolites, can relax precontracted strips. COX inhibition up-regulates the lipoxygenase metabolic pathway, accounting for the ACh contractile effect. At an intermediate resting tension, NO production is present, but COX inhibition reveals a lipoxygenase-dependent, ACh-induced contraction. At high resting tension, NO synthesis predominates and COX metabolites influence ACh-induced relaxation marginally.

Lack of nitric oxide- and guanosine 3':5'-cyclic monophosphate-dependent regulation of alpha-thrombin-induced calcium transient in endothelial cells of spontaneously hypertensive rat hearts.

While the expression and/or activity of endothelial nitric oxide synthase (eNOS) has been characterized in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rat (WKY) hearts, in coronary endothelial cells (ECs) from both strains, the effect of NO on intracellular calcium concentration ([Ca(2+)](i)) is still unknown. Coronary microvascular ECs were isolated from SHR and WKY and characterized. Immunocytochemistry and Western blot analysis showed that eNOS was similarly expressed in ECs from both strains. Measuring [Ca(2+)](i) by imaging analysis of fura-2-loaded cells, we demonstrated that alpha-thrombin (3-180 U l(-1)) induced a superimposable dose-dependent calcium transient in ECs from both strains. In WKY ECs, S-nitroso-N-acetyl-DL-penicillamine (SNAP) dose-dependently (10 - 100 microM) and 0.1 microM atrial natriuretic factor (ANF) reduced the maximum and the decay time of alpha-thrombin-induced calcium transient. The inhibitory effects of SNAP and ANF were prevented by blocking cyclic GMP-dependent protein kinase. Non selective eNOS inhibitors prolonged the decay time of alpha-thrombin-induced calcium transient, while the selective inducible NOS inhibitor 1400 W was ineffective. SNAP (100 microM) and 0.1 microM ANF increased cyclic GMP content up to 22.9 and 42.3 fold respectively. In SHR ECs, alpha-thrombin-induced calcium transient was not modified by SNAP, ANF or eNOS inhibition. SNAP (100 microM) and 0.1 microM ANF increased cyclic GMP content up to 9. 3 and 51 fold respectively. In WKY ECs, SNAP dose-dependently (10 - 100 microM) reduced also bradykinin-induced calcium transient, while in SHR ECs was ineffective. We concluded that in SHR ECs, the cyclic GMP-dependent regulation of calcium transient is lost.

Semicarbazide-sensitive amine oxidase activity in the human heart.

A semicarbazide-sensitive amine oxidase with affinity for benzylamine (Bz.SSAO) was found in the human heart. This enzymatic activity has a K(m) of 278 +/- 35.3 microM and a V(m) of 114.7 +/- 14.7 nmol mg-1 min-1 (mean +/- SE of eight hearts) for benzylamine and is strongly inhibited by 1 mM histamine and by B24, a specific inhibitor of Bz.SSAO. No diamine oxidase activity was found in the human heart. The levels of MAo and B were assayed: MAO A showed a K(m) of 137.1 +/- 16.2 microM and a V(m) of 10.4 +/- 2.5 nmol mg-1 min-1 for serotonin; MAo B had a K(m) of 9.9 +/- 1.6 microM and V(m) of 4.3 +/- 1.1 nmol mg-1 min-1 for beta-phenylethylamine (mean +/- SE of seven hearts). The human heart has high MAO B activity and Bz.SSAO with histaminase activity.

Monoamine oxidase and semicarbazide-sensitive amine oxidase activities in isolated cardiomyocytes of spontaneously hypertensive rats.

In the isolated cardiomyocytes of spontaneously hypertensive rats (SHR, 3 months old) MAO A and B activities were significantly increased compared to the myocytes in the hearts of age-matched Wistar-Kyoto rats. This increase was not associated with cardiac hypertrophy in these young animals, but might represent an early event in the development of hypertrophy. A semicarbazide-sensitive amine oxidase (SSAO) activity was found in cardiomyocytes. This activity showed a high affinity for benzylamine (Km 5-6 microM) and was not inhibited by 10(-4) M pargyline and 10(-5) M deprenyl, but was largely inhibited by 10(-4) M B24 (3,5-diethoxy-4-aminomethylpyridine), a specific inhibitor of semicarbazide-sensitive amino oxidase with high affinity for benzylamine. The SSAO enzyme of rat cardiomyocytes is a copper-amine oxidase and has a crossreactivity with the antibodies raised against pure pig plasma benzylamine oxidase. In the cardiomyocytes of 3-month old SHR rats the level of this enzymic activity is not significantly increased.