Salvatore Milano

Prostaglandin E2 regulates inducible nitric oxide synthase in the murine macrophage cell line J774

We have evaluated the role of prostaglandin E2 (PGE2) in the synthesis of nitric oxide (NO) by the activation of the inducible form of nitric oxide synthase (NOS) in the murine macrophage cell line, J774, stimulated with different doses of lipopolysaccharide (LPS). The stimulation of the J774 line with suboptimal doses of LPS (0.1 microgram/mL) caused a production of endogenous PGE2 that was capable of stimulating NOS activity inducing an increase in the NO synthesis, as attested by the fact that cyclooxygenase enzyme inhibitor, indomethacin, significantly reduced NO secretion. On the contrary, a higher dose of LPS (1 microgram/mL) produced high levels of PGE2 that reduced the levels of NOS and, subsequently, NO production. Experiments carried out with exogenous PGE2 indicated that concentrations between 1 and 10 ng/mL are able to stimulate the expression of NOS and the release of NO, while higher concentrations (> 50 ng/mL) are inhibitory. Furthermore, our data indicate that there is a network of interaction which involves NO, PGE2, and tumor necrosis factor. High levels of PGE2 inhibited TNF alpha secretion, which in turn could exert inhibitory effects on NO synthesis.

Tension-free hernia repair is associated with an increase in inflammatory response markers against the mesh ☆

BACKGROUND The purpose of this study was to evaluate the involvement of inflammatory mediators in patients undergoing Lichtenstein tension-free hernioplasty (LH) using polypropylene prosthetic materials or conventional Bassini hernia repair (BH). METHODS Thirty patients male with unilateral inguinal hernia without complications or recurrence were included in this study. Randomly, patients underwent LH or BH. Peripheral venous bloods samples were collected 24 hours prior to surgery and then 6, 24, 48 and 168 hours postoperatively. RESULTS We present evidences that LH patients showed a higher increased serum level of fibrinogen, C-reactive protein, alpha-1-antitrypsin, and interleukin-6 than BH patients. Postoperative visual analogue scales for pain were reduced on mobilization for patients undergoing LH compared with BH. Neutrophils were significantly increased only in LH compared with baseline. Ceruloplasmin, transferrin, and albumin levels were unmodified after BH or LH. CONCLUSIONS In conclusion our data show that although LH induces less pain and more rapid postoperative recovery, it is associated with an higher inflammatory response compared with BH, likely due to polypropylene mesh.

Estrogen regulates cytokine production and apoptosis in PMA-differentiated, macrophage-like U937 cells

We have investigated the effects of sex steroids, estradiol (E2), and testosterone (T) on the synthesis of tumor necrosis factor alpha (TNF‐α) and interleukin‐10 (IL‐10) in phorbol‐myristate‐acetate (PMA)‐differentiated human monoblastic U937 cells. The ability of both hormones to modulate the viability and programmed cell death of macrophage‐like PMA‐differentiated U937 cells was also inspected. E2 increased TNF‐α synthesis, whereas T had no effect on the production of this cytokine. The combination of E2 and its antagonist tamoxifen or ICI‐182,789 completely abolished the induction of TNF‐α, while combination of T and its antagonist Casodex (CSDX) did not significantly affect TNF‐α production by U937 cells. Exposure of cells to E2 resulted in a dose‐dependent decrease of IL‐10 synthesis, while again T did not show any detectable effect. In addition, E2 induced a significant increase of apoptosis in macrophage‐like U937 cells and this increase was inhibited by the simultaneous addition of either tamoxifen or ICI‐182. In contrast, T alone or in combination with CSDX did not modify apoptotic rates of U937 cells. This evidence, taken together, suggests that estrogens, but not androgens, exert a pro‐inflammatory action through the modulation of TNF‐α and IL‐10, and regulate the immune effector cells by the induction of programmed cell death. J. Cell. Biochem. 90: 187–196, 2003.

Anti-inflammatory effects of chemically modified tetracyclines by the inhibition of nitric oxide and interleukin-12 synthesis in J774 cell line

We investigated the effects of chemically modified tetracyclines (CMTs) on the production of nitric oxide (NO) and on the synthesis of some cytokines: tumour necrosis factor alpha (TNF-alpha), interleukin(IL)-10 and IL-12 in lipopolysaccharide (LPS)-treated J774 cell line. Furthermore, we studied the ability of these drugs to modify the viability in LPS-stimulated J774 macrophages. CMTs decreased, in a dose-dependent manner, inducible NO synthase (iNOS) activity and, consequently, nitrite formation in J774 cultures. The CMT-induced decrease in NO production is due to the inhibition of enzyme activity rather than to a direct effect on enzyme expression. The absence of the inhibition in mRNA accumulation indicates that the inhibiting activity is mainly post-transcriptional. CMTs were unable to modulate TNF-alpha and IL-10 synthesis and they were not effective in modifying the transcription of relative mRNA in J774 macrophages. On the contrary, IL-12 mRNA expression was significantly increased by CMT-1 and CMT-8 with LPS activation. Since IL-12 protein secretion was inhibited by CMTs, these compounds interfere in the blocking of post-transcriptional events. The studies on cell viability showed that various CMTs induced a dose-dependent decrease in J774 macrophage viability. The cytotoxic activity was present even though NO production was inhibited by CMTs. These compounds appear to be able to activate apoptosis in aNO-independent way. Altogether, these results indicate that CMTs can exert anti-inflammatory effects by inhibiting NO synthesis, and they are able to modify cell viability by exerting a strong apoptotic activity.

Anti-inflammatory effects of annexin-1: stimulation of IL-10 release and inhibition of nitric oxide synthesis.

Annexin-1 (ANX-1) is an anti-inflammatory protein induced by glucocorticoids. Like glucocorticoids, ANX-1 and derived peptides inhibit eicosanoid synthesis, block leukocyte migration and induce apoptosis of inflammatory cells. Cytokines may possess either pro-inflammatory, i.e. interleukin(IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-12 or anti-inflammatory properties, i.e. IL-4, IL-10. The experiments described in the present study have been performed to answer the question whether the anti-inflammatory action of ANX-1 may be mediated, at least in part, by the release of IL-10. In macrophage (J774) cell line cultures primed with lipolysaccharide (LPS), recombinant ANX-1 stimulated IL-10 release in a dose- and time-dependent manner. In the same cells, the protein and its derived N-terminal peptide (amino acids 2-26) dose-dependently inhibited the release of nitric oxide (NO). Furthermore, both the whole protein and the peptide down-regulated the mRNA expression of the inducible nitric oxide sythase (iNOS). The peptide was also able to inhibit the expression of IL-12 mRNA. These results suggest that some of the anti-inflammatory effects of ANX-1 may be mediated by the release of IL-10, which, in turn, inhibits iNOS mRNA expression and, hence, NO release. In addition, ANX-1-stimulated IL-10 release may also be responsible for the inhibition of IL-12 mRNA expression and, consequently, IL-12 synthesis.

Visceral adiposity index (VAI) is predictive of an altered adipokine profile in patients with type 2 diabetes.

Aims Although there is still no clear definition of “adipose tissue dysfunction” or ATD, the identification of a clinical marker of altered fat distribution and function may provide the needed tools for early identification of a condition of cardiometabolic risk. Our aim was to evaluate the correlations among various anthropometric indices [BMI, Waist Circumference (WC), Hip Circumference (HC), Waist/Hip ratio (WHR), Body Adiposity Index (BAI) and Visceral adiposity Index (VAI)] and several adipocytokines [Visfatin, Resistin, Leptin, Soluble leptin receptors (sOB-R), Adiponectin, Ghrelin, Adipsin, PAI-1, vascular endothelial growth factor (VEGF), Hepatocyte growth factor (HGF) TNF-α, hs-CRP, IL-6, IL-18] in patients with type 2 diabetes (DM2). Materials and Methods Ninety-one DM2 patients (age: 65.25±6.38 years; 42 men and 49 women) in stable treatment for the last six months with metformin in monotherapy (1.5–2 g/day) were cross-sectionally studied. Clinical, anthropometric, and metabolic parameters were evaluated. Serum adipocytokine levels were assayed with Luminex based kits. Results At the Pearson’s correlation, among all the indices investigated, VAI showed a significant correlation with almost all adipocytokines analyzed [Visfatin, Resistin and hsCRP (all p<0.001); Adiponectin, sOb-R, IL-6, IL-18, HGF (all p<0.010); Ghrelin and VEGF (both p<0.05)]. Through a two-step cluster analysis, 55 patients were identified with the most altered adipocytokine profile (patients with ATD). At a ROC analysis, VAI showed the highest C-statistic [0.767 (95% CI 0.66–0.84)] of all the indices. Conclusions Our study suggests that the VAI, among the most common indexes of adiposity assessment, shows the best correlation with the best known adipocytokines and cardiometabolic risk serum markers. Although to date we are still far from clearly identifying an ATD, the VAI would be an easy tool for clearly mirroring a condition of cardiometabolic risk, in the absence of an overt metabolic syndrome.

Identification of epitopes of Mycobacterium tuberculosis 16-kDa protein recognized by human leukocyte antigen-A*0201 CD8+ T lymphocytes

CD8(+) T cells could make an important contribution to protection against tuberculosis (TB), but the antigenic determinants recognized in the context of major histocompatibility complex class I molecules remain ill defined. Our aim was to identify nonamer peptides derived from the acr/16-kDa antigen. Two immunogenic peptides (p21-29 and p120-128) were identified by their ability to elicit cytotoxic CD8(+) T cells from juvenile patients recovering from TB. Epitope-specific recognition was demonstrated by the lysis of both Mycobacterium tuberculosis-infected and peptide-pulsed macrophages, the release of cytotoxic granules, and interferon-gamma and tumor necrosis factor-alpha production. CD8(+) T cell responses to p21-29 and p120-128 were detected ex vivo in freshly isolated peripheral blood mononuclear cells from patients with TB but not in those from healthy control subjects. Our data suggest that these antigenic peptides can play a critical role in effective immunity against mycobacterial infection and TB.

Tetracycline inhibits the nitric oxide synthase activity induced by endotoxin in cultured murine macrophages

Here we investigate the effects of tetracycline base and of a semi-synthetic tetracycline derivative, doxycycline, on the induction of inducible nitric oxide synthase and, hence, on the production of nitric oxide (NO) by lipopolysaccharide in J774 macrophage cultured in vitro. The treatment of J774 line with tetracycline base (6.25-250 microM) or doxycycline (5-50 microM) dose-dependently decreased the lipopolysaccharide-stimulated (1 microg/ml) inducible NO synthase activity and, consequently, nitrite formation. For instance, the inhibition was 70% for tetracycline base at 250 microM and 68% for doxycycline at 50 microM. The inhibitory effect of tetracyclines was due neither to a reduction in the viability of the cells, studied as colorimetric 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay, nor to an indiscriminate inhibition of total protein synthesis, but to a specific decrease in inducible NO synthase protein content in the cells, as attested by the significant reduction of the expression of inducible NO synthase, assayed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. However, no effect of tetracyclines on inducible NO synthase mRNA accumulation could be demonstrated in lipopolysaccharide-stimulated macrophage line, suggesting that the inhibitory effect of tetracyclines on NO synthesis involves post-transcriptional events. The reduction in lipopolysaccharide-stimulated nitrite accumulation produced by tetracyclines was significantly less when they were applied 6 h after lipopolysaccharide and absent 12 h after lipopolysaccharide, indicating that tetracyclines modify an early event in inducible NO synthase activation operating after mRNA transcription. The findings presented in this study indicate that the modulation of NO synthesis is another possible pathway by which tetracyclines may function as anti-inflammatory compounds.

Effects of chemically modified tetracyclines (CMTs) in sensitive, multidrug resistant and apoptosis resistant leukaemia cell lines

Recently discovered chemically modified tetracyclines (CMTs) have shown in vitro and in vivo anti‐proliferative and anti‐tumour activities. Here, we evaluated in vitro the anti‐proliferative and apoptotic activity of six different dedimethylamino chemically modified tetracyclines (CMT‐1, CMT‐3, CMT‐5, CMT‐6, CMT‐7 and CMT‐8) in sensitive and multidrug resistant myeloid leukaemia cells (HL60 and HL60R) in vitro. Three of these compounds (CMT‐5, CMT‐6, CMT‐7) showed low cytotoxic activity both in sensitive and in resistant cells, CMT‐3 was endowed with a high anti‐proliferative activity only in sensitive cells and was moderately effective as apoptosis inducing agent, with an activity similar to that shown by doxycycline. On the contrary, CMT‐1 and CMT‐8 were very effective as programmed cell death inducing agents. The apoptotic pathway activated by these compounds involved the activation of caspases, especially caspase‐9 and, for CMT‐1, also the activation of Fas. Interestingly CMT‐8, but not CMT‐1, was able to induce apoptosis in multidrug resistant HL60R and in Fas‐ligand resistant HUT78B1 cell lines. These properties, together with others previously described (e.g. anti‐metastatic and anti‐osteolytic activities), suggest that CMT‐8 may have important applications in the clinical management of cancer. The comparative analysis of structure‐activity relationship of CMT‐8 and doxycycline suggests that the C‐5 hydroxy moiety may play an important role in conferring activity in multidrug resistant cells. These findings appear to support the hypothesis that CMT‐8 may represent an interesting lead for the development of a new class of potent apoptosis inducer agents active in multidrug resistant and Fas‐ligand resistant malignancies.

CD4+ CCR5+ and CD4+ CCR3+ lymphocyte subset and monocyte apoptosis in patients with acute visceral leishmaniasis

The potential involvement of apoptosis in the pathogenesis of visceral leishmaniasis (VL) was examined by studying spontaneous and Leishmania antigen (LAg)‐induced apoptosis using cryopreserved peripheral blood mononuclear cells (PBMC) of Sicilian patients with VL. Results indicate that monocytes and T lymphocytes from acute VL patients show a significantly higher level of apoptosis compared with that observed in healed subjects. The percentage of apoptotic cells was higher in monocytes than in T lymphocytes. T cells involved in programmed cell death (PCD) were mainly of the CD4+ phenotype. In particular, the T helper 1‐type (Th1) subset, as evaluated by chemokine receptor‐5 (CCR5) expression, is involved in this process. Cell death in Th1‐type uses a CD95‐mediated mechanism. Furthermore, Th1‐type CCR5+ cells are prone to cell suicide in an autocrine or paracrine way, as attested by enhanced expression of CD95L in acute VL patients. The reduction in Th1‐type cells by apoptosis was confirmed by the decrease in interferon‐γ secretion. In conclusion, apoptosis of monocytes, CD4+ and CD4+ CCR5+ T cells could be involved in the failure of cell mediated immunity that is responsible for severe immune‐depression in VL.